Binding constructs and methods for use thereof

ABSTRACT

The invention relates to novel binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a wild-type IgG, IGA or IgE hinge-acting region, i.e., IgE CH2, region polypeptide or a mutant IgGI hinge region polypeptide having either zero, one or two cysteine residues, and immunoglobulin CH2 and CH3 domains, and that are capable of ADCC and/or CDC while occurring predominantly as polypeptides that are compromised in their ability to form disulfide-linked multimers. The fusion proteins can be recombinantly produced at high expression levels. Also provided are related compositions and methods, including cell surface forms of the fusion proteins and immunotherapeutic applications of the fusion proteins and of polynucleotides encoding such fusion proteins.

All applications from which this application takes priority areincorporated herein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 910180_(—)40102USPCa_SEQUENCE_LISTING.txt. Thetext file is 1304 KB, was created on Mar. 20, 2009, and is beingsubmitted electronically via EFS-Web.

FIELD OF THE INVENTION

The present invention relates generally to compounds having variousutilities including uses for research, diagnostics, and therapy, forexample, immunotherapy. Compounds of the invention includeimmunologically active proteins and protein conjugates. Such proteinsinclude recombinant or engineered binding proteins such as, for example,binding domain-immunoglobulin fusion proteins, which may include singlechain Fv-immunoglobulin fusion proteins and compounds containing singlechain Fv-immunoglobulins. The present invention also relates tocompositions and methods for treating conditions, diseases and disordersthat would improved, eased, or lessened from the administration of, forexample, polypeptide and/or nucleic acid constructs of the invention,including, for example, malignant conditions and B cell disorders,including diseases characterized by autoantibody production and/orinflammation.

BACKGROUND OF THE INVENTION

The immune system is one of the most complex of the body's manyintricate systems. A vast and complicated arrangement made up of manydifferent types of cells and involving many different kinds ofmolecules, the human immune system allows the body to respond to foreigninvaders such as bacteria, viruses, and other infectious agents, as wellas foreign material such as pollen. In general, the human immune systemis divided into two main parts, antibody-mediated immunity (also called“humoral” or “circulating” immunity) and cell-mediated immunity, both ofwhich are managed by lymphocytes. Lymphocytes are one of the five kindsof white blood cells (leukocytes) circulating in the blood. There areseveral kinds of lymphocytes, each with different functions to perform.The most common types of lymphocytes are B lymphocytes (B cells), whichare responsible for making antibodies, and T lymphocytes (T cells).Cells of the immune system not only include T cells and B cells, butalso Natural Killer Cells, granulocytes (or polymorphonuclear (PMN)leukocytes), macrophages, and dendritic cells. The humoral system ismanaged by B cells with help from T cells and deals with infectiousagents in the blood and tissues of the body. The cell-mediated system ismanaged by T cells and deals with cells of the body that have beeninfected.

An antigen is a substance, usually macromolecular, that stimulates orinduces an immune response. Because of its complex macromolecularstructure, a single microorganism consists of multiple antigens (e.g.,surface structures such as cell wall components, fimbriae, flagella,etc., or extracellular proteins, such as toxins or enzymes produced bythe microorganism). The coat proteins and some of the envelope proteinsof animal viruses are also usually antigenic. A host is generally ableto respond specifically to antigens that come into contact withcomponents of its immune system. Both the antibody-mediated immunity andcell-mediated immunity systems involve complex interrelationships thatallow them to mount immune reactions to almost any antigen. In otherwords, the immune system is able to recognize foreign substances(antigens) that stimulate the system to produce antibody-mediatedimmunity, cell-mediated immunity, or both.

The immune system complex is constituted by a variety of different celltypes and organs disseminated throughout the body. These include theprimary and secondary lymphoid organs. The primary lymphoid organs arethe bone marrow and the thymus. All the cells of the immune system areinitially derived from the bone marrow in a process calledhematopoiesis. During hematopoiesis bone marrow-derived stem cellsdifferentiate into either mature cells of the immune system (“B” cells)or into precursors of cells that migrate out of the bone marrow tomature in the thymus (“T” cells). In addition to red blood cells,platelets, and B cells, the bone marrow also produces Natural Killercells, granulocytes, and immature thymocytes. The function of the thymusis to produce mature T cells. Immature thymocytes, also known asprothymocytes, leave the bone marrow and migrate into the thymus wherethey mature and are then released into the bloodstream. The immunesystem complex also includes secondary lymphoid organs, e.g., thespleen, the lymph nodes, etc., as well as a circulatory system that isseparate from blood vessels.

The spleen, made up of B cells, T cells, macrophages, dendritic cells,Natural Killer cells, and red blood cells, is an immunologic filter ofthe blood. Migratory macrophages and dendritic cells capture antigensfrom blood that passes through the spleen. Migratory macrophages anddendritic cells also bring antigens to the spleen via the bloodstream.An immune response is initiated in the spleen when macrophages ordendritic cells present the antigen to the appropriate B or T cells, andB cells become activated and produce large amounts of antibody.

Lymphatic vessels and lymph nodes are the parts of a special circulatorysystem that carries lymph. Lymph is a transparent fluid containing whiteblood cells, chiefly lymphocytes. Lymph bathes the tissues of the body,and is then collected in lymphatic vessels. Lymph nodes dot a network oflymphatic vessels and, when afferent lymph ducts bring lymph-containingantigens into the node, function as an immunologic filter for lymph.Composed mostly of T cells, B cells, dendritic cells, and macrophages,the lymph nodes drain fluid from most tissues. Antigens are filtered outof the lymph in the lymph node before the lymph is returned to thecirculation. Macrophages and dendritic cells that capture antigens alsopresent these foreign materials to T and B cells in the lymph nodes,resulting in the stimulation of B cells to develop there intoantibody-secreting plasma cells. Antibodies leave the lymph node by theefferent ducts that empty into the blood stream. Lymphocytes can alsoleave the node by the efferent duct and travel to other sites in thelymphatic system or enter into the blood circulation. A singlelymphocyte completes a circuit through the circulating blood andlymphatic systems once every 24 hours.

Tonsils, adenoids, Peyer's patches, and the appendix are also lymphoidtissues. Peyer's patches (masses of lymphocytes) are similar to thetonsils and are found throughout the body, especially in the mucouslinings of the digestive and respiratory tracts. It is the function ofthe phagocytic cells found in Peyer's patches and other lymphaticaggregate follicles to defend the body against, for example,inadequately digested food particles crossing the gut wall and enteringthe blood, and to attack unwanted foreign invaders while they are stillin the bowel.

The major function of B cells is the production of antibodies inresponse to foreign proteins of bacteria, viruses, and tumor cells. Tcells are usually divided into two major groups, namely, the cytotoxic Tlymphocytes (“Tc” cells or CTLs) and the helper T cells (“Th” cells or Thelper cells). Th cells, also referred to as CD4+ T cells, function toaugment or potentiate immune responses by the secretion of specializedfactors that activate other white blood cells to fight off infection.They enhance the production of antibodies by B cells. Tc cells, alsocalled CD8+ T cells, can directly kill certain tumor cells,viral-infected cells, and sometimes parasites. Tc cells are alsoimportant in down-regulation of immune responses. Both types of T cellsoften depend on the secondary lymphoid organs (the lymph nodes andspleen) as sites where activation occurs, but they are also found inother tissues of the body, including the liver, lung, blood, andintestinal and reproductive tracts.

Natural Killer cells, often referred to as NK cells, represent anothertype of lymphocyte and are similar to the Tc cell subset. They functionas effector cells that directly kill certain tumors such as melanomasand lymphomas, and viral-infected cells. They are called “natural”killers because, unlike cytotoxic T cells, they do not need to recognizea specific antigen before carrying out their function. While NK cells,unlike the Tc cells, kill their targets without prior activation in thelymphoid organs, NK cells activated by Th cell secretions will killtumor or viral-infected targets more effectively. NK cells target tumorcells and protect against a wide variety of infectious microbes. Inseveral immunodeficiency diseases, including AIDS, Natural Killer cellfunction is abnormal. Natural Killer cells may also contribute toimmunoregulation by secreting high levels of influential lymphokines.

Some NK cells have surface receptors (FcγRIII, also called CD16) for theFc portion of the IgG antibody. They bind to target cells throughreceptors for the Fc portion of an antibody that has reacted withantigen on a target cell. This type of cell-mediated immunity is calledantibody-dependent cell-mediated cytotoxicity (ADCC). NK cells may alsohave receptors for the C3 component of complement, another immunedefense system, and therefore recognize cells that are coated with C3 astargets. ADCC is thought to be an important defense against a variety ofparasitic infections caused, for example, by protozoa and helminths.

Although small lymphocytes look identical, they can be distinguished bymolecules carried on their cell surface. Not only do such markersdistinguish between B cells and T cells, they distinguish among varioussubsets of cells that behave differently. Every mature T cell, forinstance, carries a marker known as T3 (or CD3). In addition, mosthelper T cells carry a T4 (CD4) marker, a molecule that recognizes ClassII major histocompatibility complex (“MHC”) antigens. A molecule knownas T8 (CD8), which recognizes Class I MHC antigens, is found on manysuppressor/cytotoxic T cells.

Another group of white blood cells collectively referred to asgranulocytes, or polymorphonuclear leukocytes (PMNs), is composed ofthree cell types. These cells, neutrophils, eosinophils, and basophilsare important in the removal of bacteria and parasites from the body.Neutrophils migrate through capillary walls and into infected tissuewhere they kill invaders (e.g., bacteria) and then engulf the remnantsby phagocytosis. Eosinophils are cytotoxic, releasing the contents oftheir granules on an invader. Basophils leave the blood and accumulateat the site of an infection or other inflammation and discharge thecontents of their granules, releasing a variety of mediators such ashistamine, serotonin, prostaglandins and leukotrienes that, for example,increase blood flow to the area. Mediators released by basophils alsoplay an important part in some allergic responses such as hay fever andanaphylactic responses to insect stings.

Monocytes are large phagocytic white blood cells released from the bonemarrow into the blood circulation. When a monocyte enters tissue, itdevelops into a macrophage. Macrophages are also large, phagocytic cellsthat engulf foreign material (antigens) that enter the body, as well asdead and dying cells of the body. Macrophages are important in theregulation of immune responses, and are often referred to as scavengers,or antigen-presenting cells (APCs) because they pick up and ingestforeign materials and present these antigens to other cells of theimmune system such as T cells and B cells. This is one of the importantfirst steps in the initiation of an immune response. Stimulatedmacrophages exhibit increased levels of phagocytosis and also secreteInterleukin-1 (IL-1), a product that helps to activate B cells and Tcells.

Dendritic cells also originate in the bone marrow and function as APCs.They are usually found in the structural compartment of lymphoid organssuch as the thymus, lymph nodes and spleen, but are also found in thebloodstream and other tissues. It is believed that dendritic cellscapture antigen or bring it to the lymphoid organs where an immuneresponse is initiated.

Important features of the immunological system relevant to host defenseand/or immunity to pathogenic microorganisms include specificity,memory, and tolerance. It is understood, for example, that an antibodyor reactive T cell will react specifically with the antigen that inducedits formation; it will not react with other antigens. Generally, thisspecificity is of the same order as that of enzyme-substrate specificityor receptor-ligand specificity, although cross-reactivity is possible.The specificity of the immune response is explained by clonal selection.During the primary immune response, a specific antigen selects apre-existing clone of specific lymphocytes and stimulates itsactivation, proliferation and differentiation. It is also understoodthat once the immune system has responded to produce a specific type ofantibody or reactive T cell, it is capable of producing more of theantibody or activated T cell more rapidly and in larger amounts; this iscalled the secondary (or memory) response. It is also recognized that ananimal generally does not undergo an immunological response to its own(potentially-antigenic) components. The animal is said to be tolerant,or unable to react to its own potentially antigenic components. Thisensures that under normal conditions, an immune response to “self”antigens (called an autoimmune response) does not occur. Tolerance isbrought about in a number of ways, but in essence the immune system isable to distinguish “self” components from “non-self' (foreign)antigens; it will respond to “non-self” but not to “self”. Sometimes inan animal, tolerance can be “broken”, which may result in an autoimmunedisease.

The biological activities of the antibody-mediated and cell-mediatedimmune responses are different and vary from one type of infection toanother. There are several classes or types of antibodies (andsubclasses of various types) involved in antibody-mediated immunity. Allof the classes of antibodies that are produced in response to a specificantigen react stereochemically with that antigen and not with other(different) antigens. The host has the genetic capacity to producespecific antibodies to thousands of different antigens, but does not doso until there is an appropriate (specific) antigenic stimulus. Due toclonal selection, the host produces only the homologous antibodies thatwill react with that antigen which, as noted above, are found in blood(plasma), lymph, and many extravascular tissues. Once theantibody-mediated immunity response occurs following interaction of Blymphocytes with antigen and their differentiation intoantibody-secreting plasma cells, the secreted antibody binds to theantigen which, in turn, results in its neutralization or eliminationfrom the body.

Cell-mediated immunity, on the other hand, is mediated by specificsubpopulations of T-lymphocytes called effector T cells that exist inprecursor form as “resting T cells” (pT cells). These cells bearreceptors for specific antigens and recognize these antigens on thesurfaces of other cells. Stimulation with that antigen results in T cellactivation. T cells enlarge, enter into a mitotic cycle, reproduce anddevelop into effector T cells whose activities are responsible for thistype of immunity. They also develop into clones of identical reactive Tcells called memory T cells. As noted above, most of the T cells in thebody belong to one of two subsets and are distinguished by the presenceon their surface of one or the other of two glycoproteins designated CD4and CD8. Which of these molecules is present determines the types ofcells to which the T cell can bind. T cells bearing CD8 (CD8′ T cells)always recognize antigen in association with Class I MHC proteins andtypically function as cytotoxic T cells. Almost all the cells of thebody express Class I MHC molecules. T cells bearing CD4 (CD4′ T cells)always recognize antigens in association with Class II MHC proteins onthe surfaces of other cells. Only specialized antigen-presenting cellsexpress Class II MHC molecules, including dendritic cells, phagocyticcells such as macrophages, and B cells. CD4′ T lymphocytes generallyfunction as T helper cells.

T helper cells, which include Th1 cells and Th2 cells, respond toantigen with the production of lymphokines Th1 and Th2 cells can bedistinguished based on their lymphokine profiles. Like all T cells, Thcells arise in the thymus. When they are presented with an antigen byantigen-presenting dendritic cells they begin to proliferate and becomeactivated. There are two kinds of dendritic cell, DC1 cells (descendedfrom monocytes) and DC2 cells (which appear to be derived fromlymphocytes).

Th1 cells (inflammatory Th1 cells involved in the elimination ofpathogens residing intracellularly in vesicular compartments) areproduced when DC1-type dendritic cells present antigen to the T cellreceptor for antigen (TCR) and secrete Interleukin 12 (IL-12). Thisparacrine stimulation activates Th1 cells to secrete their ownlymphokines, in particular, Tumor-Necrosis Factor-beta (TNF-β) (alsoknown as lymphotoxin) and Interferon-gamma (IFN-γ). These lymphokinesstimulate macrophages to kill bacteria they have engulfed byphagocytosis and they recruit other leukocytes to the site producinginflammation. Th1 cells are essential for cell-mediated immunity and forcontrolling intracellular pathogens such as, for example, Listeria andMycobacterium tuberculosis.

Th2 cells (“true” helper Th2 cells, which are required for antibodyproduction by B cells) are produced when DC2-type dendritic cellspresent antigen to the T cell receptor for antigen and, presumably, oneor more paracrine stimulants. The major lymphokines secreted by Th2cells are Interleukin 4 (IL-4), which stimulates class-switching in Bcells and promotes their synthesis of IgE antibodies, acts as apositive-feedback device promoting more pre-Th cells to enter the Th2pathway, and blocks expression of the IL-12 receptor thereby inhibitingpre-Th cells in the thymus from entering the Th1 pathway. IL-4 alsocauses B cells to proliferate and differentiate into antibody-secretingplasma cells and memory B cells. IL-4 activates only B cells in thevicinity which themselves have bound the antigen, and not others, so asto sustain the specificity of the immune response. Th2 cells alsoproduce Interleukin 5 (IL-5, which attracts and activates eosinophils),Interleukin 10 (IL-10, which inhibits IL-12 production by DCs andprevents maturation of pre-Th cells to Th1 cells), and Interleukin 13(IL-13, which also promotes the synthesis of IgE antibodies).

Activation of the Th2 cell also causes it to begin to produceInterleukin 2 (IL-2), and to express a membrane receptor for IL-2. Thesecreted IL-2 autostimulates proliferation of Th2 cells. For example,IL-2 binds to IL-2 receptors on other T cells (which have bound theantigen) and stimulates their proliferation. In addition to IL-2,stimulated Th2 cells also produce IFN-γ and Interleukin 6 (IL-6), whichmediate various aspects of the immune response. IFN-γ activates NaturalKiller cells to their full cytolytic potential, and is an activator ofmacrophages and thus increases their antitumor activities. If themacrophages are infected by intracellular parasites, it activatesmacrophages, which in turn destroy the parasites. IFN-γ also reinforcesthe antitumor activities of the cytotoxic lymphocytes, increases thenonspecific activities of NK-cells, and is one of the factors thatcontrols the differentiation of B cells and increases the secretion ofimmunoglobins. IL-6 stimulates several types of leukocytes, as well asthe production of Acute Phase Proteins in the liver. It is particularlyimportant in inducing B cells to differentiate into antibody forming(plasma) cells. Thus, Th2 cells provide help for B cells and areessential for antibody-mediated immunity.

Cytotoxic T lymphocytes are able to kill cells that show a new orforeign antigen on their surface (for example, virus-infected cells, ortumor cells, or transplanted tissue cells). The CD8⁺ CTLs also come intwo subsets: Tc1 that, like Th1 cells, secrete IFN-γ, and Tc2 that, likeTh2 cells, secrete IL-4.

The cell-mediated immunity response also plays a role in destruction oftumor cells and in rejection of tissue transplants in animals. A majorproblem in tissue transplantation is rejection, which is often based oncell-mediated immunity response to “foreign” cells (because they are nota perfect antigenic match). Because tumor cells contain specificantigens not seen on normal cells they also may be recognized as foreignand destroyed by the forces of cell-mediated immunity. If tumor cellsdevelop on a regular basis in animals, it may be cell-mediated immunitythat eliminates them or holds them in check. The increase in theincidence of many types of cancer (tumors) in humans with advancement ofage may be correlated with a decline in the peak efficiency of theimmune system that occurs about 25 years of age.

A summary of the types of cells involved in the expression ofcell-mediated immunity follows. Tc lymphocytes kill cells bearingforeign antigen on surface in association with Class I MHC and can killcells that are harboring intracellular parasites (either bacteria orviruses) as long as the infected cell is displaying a microbial antigenon its surface. Tc cells kill tumor cells and account for rejection oftransplanted cells. Tc cells recognize antigen-Class I MHC complexes ontarget cells, contact them, and release the contents of granulesdirectly into the target cell membrane that lyses the cell. Thlymphocytes produce lymphokines that are “helper” factors fordevelopment of B cells into antibody-secreting plasma cells. They alsoproduce certain lymphokines that stimulate the differentiation ofeffector T lymphocytes and the activity of macrophages. Th1 cellsrecognize antigen on macrophages in association with Class II MHC andbecome activated (by IL-1) to produce lymphokines including IFN-γ thatactivates macrophages and NK cells. These cells mediate various aspectsof the cell-mediated immunity response including delayed-typehypersensitivity reactions. Th2 cells recognize antigen in associationwith Class II MHC on an APC and then produce interleukins and othersubstances that stimulate specific B cell and T cell proliferation andactivity. Macrophages are important as antigen-presenting cells thatinitiate T cell interactions, development, and proliferation.Macrophages are also involved in expression of cell-mediated immunitybecause they become activated by IFN-γ produced in a cell-mediatedimmunity response. Activated macrophages have increased phagocyticpotential and release soluble substances that cause inflammation anddestroy many bacteria and other cells. Natural Killer cells arecytotoxic cells that lyse cells bearing new antigen regardless of theirMHC type and even lyse some cells that bear no MHC proteins. NK cellsare defined by their ability to kill cells displaying a foreign antigen(e.g., tumor cells) regardless of MHC type and regardless of previoussensitization (exposure) to the antigen. NK cells can be activated byIL-2 and IFN-γ, and lyse cells in the same manner as cytotoxic Tlymphocytes. Some NK cells have receptors for the Fc domain of the IgGantibody and are thus able to bind to the Fc portion of IgG on thesurface of a target cell and release cytolytic components that kill thetarget cell via antibody-dependent cell-mediated cytotoxicity.

Extracellular factors that affect cell proliferation and differentiationhave been defined as cytokines. These include the lymphokines, which areproteins produced by T-lymphocytes that have effects on thedifferentiation, proliferation, and activity of various cells involvedin the expression of cell-mediated immunity. In general, lymphokinesfunction by (1) focusing circulating leukocytes and lymphocytes into thesite of immunological encounter; (2) stimulating the development andproliferation of B cells and T cells; (3) stimulating and preparingmacrophages for their phagocytic tasks; (4) stimulating Natural Killercells; and (5) providing antiviral cover and activity. A summary ofvarious important lymphokines follows. Initially referred to aslymphocyte activation factor, IL-1 is mainly a product of macrophages,and has a variety of effects on various types of cells. It acts as agrowth regulator of T cells and B cells, and it induces other cells suchas hepatocytes to produce proteins relevant to host defense. IL-1 formsa chemotactic gradient for neutrophils and serves as an endogenouspyrogen that produces fever. Thus, IL-1 plays an important role in boththe immune responses and in the inflammatory response. IL-2 stimulatesthe proliferation of T cells and activates NK cells. IL-3 regulates theproliferation of stem cells and the differentiation of mast cells. IL-4causes B cell proliferation and enhanced antibody synthesis. IL-6 (alsoreferred to as Interferon-beta2, hybridoma growth factor, B-celldifferentiation factor, and hepatocyte stimulatory factor) has effectson B cell differentiation and on antibody production and on T cellactivation, growth, and differentiation, and probably has a major rolein the mediation of the inflammatory and immune responses initiated byinfection or injury. IL-8 is a chemotactic attractant for neutrophils.IL-13 shares many of the properties of IL-4, and is a potent regulatorof inflammatory and immune responses. IFN-γ is produced by T cells andmay be considered a lymphokine It is sometimes called “immuneinterferon” (Interferon-alpha being referred to as “leukocyteinterferon” and Interferon-beta being referred to as “fibroblastinterferon”). IFN-γ has several antiviral effects including inhibitionof viral protein synthesis in infected cells. It also activatesmacrophages and NK cells, and stimulates IL-1, IL-2, and antibodyproduction. Lymphotoxins include the Tumor Necrosis Factors. T cellsproduce TNF-beta, while TNF-alpha is produced by T cells as well asother types of cells. TNFs function to kill cells, including tumor cells(at a distance). There are several Colony Stimulating Factors (CSFs),including granulocyte macrophage colony stimulating factor (GMCSF),which cause phagocytic white cells of all types to differentiate anddivide.

The nature of the membrane receptors for antigen on B cells and T cellsis fairly well understood. Each B cell has approximately 10⁵membrane-bound antibody molecules (IgD or IgM) that correspond inspecificity to the antibody the cell is programmed to produce (thesereceptors being referred to as BCRs). CD32 (FcγRII) on B cells arereceptors for the Fc region of IgG. CD21 and CD35 on B cells arereceptors for complement components. Each T cell has about 10⁵ moleculesof a specific antigen-binding T cell receptor (a TCR) exposed on itssurface. The TCR is similar, but not identical, to an antibody. Thereare two types of T cells that differ in their TCRs, alpha/beta (αβ) Tcells and gamma/delta (γε) T cells. The TCR of alpha/beta T cells bindsa bimolecular complex displayed by a Class I MHC molecule at the surfaceof an antigen-presenting cell. As noted above, most Th cells expressCD4, whereas most Tc cells express CD8.

Both BCRs and TCRs are similar in that they are integral membraneproteins, they are present in thousands of identical copies exposed atthe cell surface, they are made before the cell ever encounters anantigen, they are encoded by genes assembled by the recombination ofsegments of DNA, they have a unique binding site that binds throughnon-covalent forces to a portion of the antigen called an epitope (orantigenic determinant) that depends on complementarity of the surface ofthe receptor and the surface of the epitope, and successful binding ofthe antigen receptor to the epitope, if accompanied by additionalsignals, results in stimulation of the cell to leave G₀ and enter thecell cycle and repeated mitosis that leads to the development of a cloneof cells bearing the same antigen receptor, i.e., a clone of cells ofthe identical specificity. BCRs and TCRs differ in their structure, thegenes that encode them, and the type of epitope to which they bind.

Induction of a primary immune response begins when an antigen penetratesepithelial surfaces. It will eventually come into contact withmacrophages or certain other classes of antigen presenting cells,including B cells, monocytes, dendritic cells, Langerhans cells, andendothelial cells. Antigens, such as bacterial cells, are internalizedby endocytosis and “processed” by APCs, then “presented” toimmunocompetent lymphocytes to initiate the early steps of theimmunological response. Processing by a macrophage (for example) resultsin attaching antigenic materials to the surface of the membrane inassociation with Class II MHC molecules on the surface of the cell. Theantigen-class II MHC complex is presented to a T-helper (Th2) cell,which is able to recognize processed antigen associated with a Class IIMHC molecule on the membrane of the macrophage. This interaction,together with stimulation by IL-1 from secreted by the macrophage, willactivate the Th2 cell.

As indicated above, B cells themselves behave as APCs. Cross-linkedantigens bound to antibody receptors on the surface of a B cell causeinternalization of some of the antigen and expression on the B cellmembrane together with Class II MHC molecules. The Th2 cell recognizesthe antigen together with the Class II MHC molecules, and secretes thevarious lymphokines that activate the B cells to becomeantibody-secreting plasma cells and memory B cells. Even if the antigencannot cross-link the receptor, it may be endocytosed by the B cell,processed, and returned to the surface in association with Class II MHCwhere it can be recognized by specific Th2 cells which will becomeactivated to initiate B cell differentiation and proliferation. In anycase, the overall B cell response leads to antibody-mediated immunity.

The antigen receptors on B cell surfaces are thought to be the specifictypes of antibodies that they are genetically programmed to produce.Hence, there are thousands of sub-populations of B cells distinguishedby their ability to produce a unique antibody molecule. B cells can alsoreact with a homologous antigen on the surface of the macrophage, orwith soluble antigens. When a B cell is bound to antigen, andsimultaneously is stimulated by IL-4 produced by a nearby Th2 cell, theB cell is stimulated to grow and divide to form a clone of identical Bcells, each capable of producing identical antibody molecules. Theactivated B cells further differentiate into plasma cells whichsynthesize and secrete large amounts of antibody, and into memory Bcells. The antibodies produced and secreted by the plasma cells willreact specifically with the homologous antigen that induced theirformation. Many of these reactions lead to host defense and toprevention of reinfection by pathogens. Memory cells play a role insecondary immune responses. Plasma cells are relatively short-lived(about one week) but produce large amounts of antibody during thisperiod. Memory cells, on the other hand, are relatively long-lived andupon subsequent exposure to antigen they become quickly transformed intoantibody-producing plasma cells.

Generation of cell mediated immunity begins when, for example, acytotoxic T cell recognizes a processed antigen associated with Class IMHC on the membrane of a cell (usually an altered self cell, butpossibly a transplanted tissue cell, or a eukaryotic parasite). Understimulation by IL-2 produced by Th2 cells, the Tc cell becomes activatedto become a cytotoxic T lymphocyte capable of lysing the cell which isshowing the new foreign antigen on its surface, a primary manifestationof cell-mediated immunity. The interaction between an antigen-presentingmacrophage and a Th cell stimulates the macrophage to produce andsecrete a Interleukin-1 that acts locally on the Th cell, stimulatingthe Th-cell to differentiate and produce its own cytokines (which mayhere be called lymphokines because they arise from a lymphocyte). Theselymphokines have various functions. IL-4 has an immediate effect onnearby B cells. IL-2 has an immediate effect on T cells as describedabove.

Leucocytes also express adhesion-promoting receptors that mediatecell-cell and cell-matrix interactions. These adhesive interactions arecrucial to the regulation of haemopoiesis and thymocyte maturation, thedirection and control of leucocyte traffic and migration throughtissues, and the development of immune and non-immune inflammatoryresponses. Several families of adhesion receptors have been identified.The leucocyte integrin family comprises three alpha-beta heterodimericmembrane glycoproteins that share a common beta subunit, designatedCD18. The alpha subunits of each of the three members, lymphocytefunction associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1) andp150, are designated CD11a, b, and c respectively. These adhesionmolecules play a critical part in the immune and inflammatory responsesof leucocytes. The leucocyte integrin family is, in turn, part of theintegrin superfamily, members of which are evolutionally, structurallyand functionally related. Another integrin subfamily found on leucocytesis the VLA group, so-called because the “very late activation antigens”VLA-1 and VLA-2 were originally found to appear late in T-cellactivation. Members of this family function mainly as extracellularmatrix adhesion receptors and are found both on haemopoietic andnon-haemopoietic cells. They play a part in diverse cellular functionsincluding tissue organization, lymphocyte recirculation and T-cellimmune responses. Another integrin subfamily, the cytoadhesins, arereceptors on platelets and endothelial cells that bind extracellularmatrix proteins. A second family of adhesion receptors is theimmunoglobulin superfamily, members of which include CD2, LFA-3, andICAM-1, which participate in T-cell adhesive interactions, and theantigen-specific receptors of T and B cells, CD4, CD8, and the MHC ClassI and II molecules. Another recognized family of adhesion receptors isthe selectins, characterized by a common lectin domain. Leucocyteadhesion molecule-1 (LAM-1), which is the human homologue of the murinehoming receptor, MEL-14, is expressed on leucocytes, while endothelialleucocyte adhesion molecule-1 (ELAM-1) and granule membrane protein(GMP-140) are expressed on stimulated endothelial cells and activatedplatelets.

Activation of an immune response requires physical cell-cell contact inaddition to cytokines. Thus, for example, development of B and T cellprecursors require intimate contact with stromal cells. At least threecritical cell-cell contact events are required for the generation ofimmune responses. The first is initial contact of a specific antigenwith a naive T cell. Because of the requirement for MHC presentation,this is an obligate cell contact event. In normal situations thecritical antigen presenting cell is the dendritic cell. In addition tothe MHC/peptide-TCR interaction there are other non-antigen specificmembrane bound ligand-receptor pairs that are important for thedendritic cell-T cell interaction. The principal one is the associationof the CD28 molecule on the T cell with either of two ligands, B7.1(CD80) and B7.2 (CD86), on the dendritic cell. These molecules aretermed accessory molecules and it is understood that the CD28 moleculedelivers an essential second signal to the T cell without which the Tcell does not become activated.

A second essential cell-cell contact is between the activated T cell andan antigen-specific B cell. Most antigens are T cell-dependent, that is,an antibody response to the antigen absolutely requires T cell help.This help is delivered both by cytokines and by cell-cell contact. Cellsbind specific antigen via surface Ig, then internalize, process, andpresent it on Class II MHC molecules. This enables them to be recognizedby T cells specific for helper epitopes from the specific antigen. Thiscell-cell interaction also requires CD28 binding to B7 on the B cell. Inaddition, a molecule called CD40 ligand or CD154, the expression ofwhich is induced upon T cell activation, binds to CD40 on B cells. CD40crosslinking promotes B cell proliferation, prevents apoptosis ofgerminal-center B cells, and promotes immunoglobulin isotype switching.The CD28-B7 and CD40-CD40L receptor ligand interactions are bothessential for the dialogue between B and T cells that causes theirmutual activation.

A third cell-cell interaction that is essential in immune responses isthe binding of activated B cells (which have migrated into a specializedstructure in lymphoid organs called germinal centers) to folliculardendritic cells (FDCs). FDCs are specialized stromal cells that holdintact, i.e., unprocessed, antigen on their surface in the form oflong-lived immune complexes. Among other molecules, FDCs express CD23,which binds to germinal center B cells via a CR2 receptor and stimulatesdifferentiation to plasma cells.

Time is required before a primary immune response is effective as a hostdefense. Antigens have to be recognized, taken up, digested, processed,and presented by APCs. A few select Th cells must react with antigen andrespond; preexisting B or T lymphocytes must encounter the antigen andproliferate and differentiate into effector cells (plasma cells or Tccells). In the case of antibody-mediated immunity, antibody level has tobuild up to an effective physiological concentration to render its hostresistant. It may take several days or weeks to reach a level ofeffective immunity, even though this immunity may persist for manymonths, or years, or even a lifetime, due to the presence of theantibodies. In natural infections, the inoculum is small, and eventhough the antigenic stimulus increases during microbial replication,only small amounts of antibody are formed within the first few days, andcirculating antibody is not detectable until about a week afterinfection.

With regard to induction of a secondary immune response, it isunderstood that on re-exposure to microbial antigens (secondary exposureto antigen), there is an accelerated immunological response, i.e., thesecondary or memory response. Larger amounts of antibodies are formed inonly 1-2 days. This is due to the activities of specific memory B cellsor memory T cells which were formed during the primary immune response.These memory cells, when stimulated by homologous antigen, “remember”having previously seen the antigen, and are able to rapidly divide anddifferentiate into effector cells. Stimulating memory cells to rapidlyproduce very high (effective) levels of persistent circulatingantibodies is the basis for giving “booster”-type vaccinations to humansand pets. Thus, following the first exposure to an antigen the immuneresponse (as evidenced by following the concentration of specificantibody in the serum) develops gradually over a period of days, reachesa low plateau within 2-3 weeks, and usually begins to decline in arelatively short period of time. When the antigen is encountered asecond time, a secondary (memory) response causes a rapid rise in theconcentration of antibody, reaching a much higher level in the serum,which may persist for a relatively long period of time. A protectivelevel of antibody may be reached by primary exposure alone, but usuallyto ensure a high level of protective antibody that persists over a longperiod of time, it is necessary to have repeated antigenic stimulationof the immune system.

An immunoglobulin molecule (abbreviated Ig), is a multimeric proteincomposed of two identical light chain polypeptides and two identicalheavy chain polypeptides (H₂L₂) that are joined into a macromolecularcomplex by interchain disulfide bonds, i.e., covalent bonds between thesulfhydryl groups of neighboring cysteine residues. There are variousclasses of human antibody proteins, each of which is produced by aspecific clone of plasma cells. Five human immunoglobulin classes aredefined on the basis of their heavy chain composition, and are namedIgG, IgM, IgA, IgE, and IgD. The IgG-class and IgA-class antibodies arefurther divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4, andIgA1 and IgA2. Intrachain disulfide bonds join different areas of thesame polypeptide chain, which results in the formation of loops that,along with adjacent amino acids, constitute the immunoglobulin domains.At the amino-terminal portion (also called the “NH₂-terminus” or the“N-terminus”), each light chain and each heavy chain has a singlevariable region that shows considerable variation in amino acidcomposition from one antibody to another. The light chain variableregion, V_(L), associates with the variable region of a heavy chain,V_(H), to form the antigen binding site of the immunoglobulin, calledthe Fv.

In addition to variable regions, each of the antibody chains has one ormore constant regions. Light chains have a single constant regiondomain. Thus, light chains have one variable region and one constantregion. Heavy chains have several constant region domains. The heavychains in IgG, IgA, and IgD antibodies have three constant regiondomains, which are designated CH₁, CH₂, and CH₃, and the heavy chains inIgM and IgE antibodies have four constant region domains, CH1, CH2, CH3and CH4. Thus, heavy chains have one variable region and three or fourconstant regions. Immunoglobulin structure and function are reviewed,for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual,Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988).

The heavy chains of immunoglobulins can also be divided into threefunctional regions: the Fd region (a fragment comprising V_(H) and CH1,i.e., the two N-terminal domains of the heavy chain), the hinge region,and the Fc region (the “fragment crystallizable” region, derived fromconstant regions and formed after pepsin digestion). The Fd region incombination with the light chain forms an Fab (the “fragmentantigen-binding”). Because an antigen will react stereochemically withthe antigen-binding region at the amino terminus of each Fab the IgGmolecule is divalent, i.e., it can bind to two antigen molecules. The Fccontains the domains that interact with immunoglobulin receptors oncells and with the initial elements of the complement cascade. Thus, theFc fragment is generally considered responsible for the effectorfunctions of an immunoglobulin, such as complement fixation and bindingto Fc receptors. Pepsin sometimes also cleaves before the third constantdomain (CH3) of the heavy chain to give a large fragment F(abc) and asmall fragment pFcb. These terms are also used for analogous regions ofthe other immunoglobulins. The hinge region, found in IgG, IgA, and IgDclass antibodies, acts as a flexible spacer, allowing the Fab portion tomove freely in space. In contrast to the constant regions, the hingedomains are structurally diverse, varying in both sequence and lengthamong immunoglobulin classes and subclasses.

For example, the length and flexibility of the hinge region varies amongthe IgG subclasses. The hinge region of IgG1 reportedly encompassesamino acids 216-231 and because it is freely flexible, the Fab fragmentscan rotate about their axes of symmetry and move within a spherecentered at the first of two inter-heavy chain disulfide bridges. IgG2has a shorter hinge than IgG1, reportedly 12 amino acid residues andfour disulfide bridges. The hinge region of IgG2 lacks a glycineresidue, it is relatively short and contains a rigid poly-proline doublehelix, stabilised by extra inter-heavy chain disulfide bridges. Theseproperties restrict the flexibility of the IgG2 molecule. IgG3 differsfrom the other subclasses by its unique extended hinge region (aboutfour times as long as the IgG1 hinge), and is reported to contain 62amino acids (including 21 prolines and 11 cysteines), forming aninflexible poly-proline double helix. In IgG3 the Fab fragments arerelatively far away from the Fc fragment, giving the molecule a greaterflexibility. The elongated hinge in IgG3 is also responsible for itshigher molecular weight compared to the other subclasses. The hingeregion of IgG4 is shorter than that of IgG1 and its flexibility isintermediate between that of IgG1 and IgG2. The flexibility of the hingeregion reportedly decreases in the order IgG3>IgG1>IgG4>IgG2. The fourIgG subclasses also differ from each other with respect to theireffector functions. This difference is related to differences instructure, including with respect to the interaction between thevariable region, Fab fragments, and the constant Fc fragment.Nevertheless, aside from glycosylation within the CH2 region, forexample, and in spite of this knowledge there are no set rules orconventions regarding means or methods to change features, includingsequences, of these subclasses of molecule to change, control, add, orremove different functions, for example, ADCC, CDC, and other functions.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. Shin et al.,1992 Immunological Reviews 130:87. The upper hinge region includes aminoacids from the carboxyl end of CH1 to the first residue in the hingethat restricts motion, generally the first cysteine residue that formsan interchain disulfide bond between the two heavy chains. The length ofthe upper hinge region correlates with the segmental flexibility of theantibody. The core hinge region contains the inter-heavy chain disulfidebridges, and the lower hinge region joins the amino terminal end of theCH2 domain and includes residues in CH2. Id. The core hinge region ofhuman IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO:515) which,when dimerized by disulfide bond formation, results in a cyclicoctapeptide believed to act as a pivot, thus conferring flexibility. Thehinge region may also contain one or more glycosylation sites, whichinclude a number of structurally distinct types of sites forcarbohydrate attachment. For example, IgA1 contains five glycosylationsites within a 17 amino acid segment of the hinge region, conferringresistance of the hinge region polypeptide to intestinal proteases,considered an advantageous property for a secretory immunoglobulin.

Conformational changes permitted by the structure and flexibility of theimmunoglobulin hinge region polypeptide sequence may also affect theeffector functions of the Fc portion of the antibody. Three generalcategories of effector functions associated with the Fc region include(1) activation of the classical complement cascade, (2) interaction witheffector cells, and (3) compartmentalization of immunoglobulins. Thedifferent human IgG subclasses vary in the relative efficacies withwhich they fix complement, or activate and amplify the steps of thecomplement cascade. See, e.g., Kirschfink, 2001 Immunol. Rev. 180:177;Chakraborti et al., 2000 Cell Signal 12:607; Kohl et al., 1999 Mol.Immunol. 36:893; Marsh et al., 1999 Curr. Opin. Nephrol. Hypertens.8:557; Speth et al., 1999 Wien Klin. Wochenschr. 111:378.

Complement-dependent cytotoxicity (CDC) is believed to be a significantmechanism for clearance of specific target cells such as tumor cells.CDC is a stream of events that consists of a series of enzymes thatbecome activated by each other in a cascade fashion. Complement has animportant role in clearing antigen, accomplished by its four majorfunctions: (1) local vasodilation; (2) attraction of immune cells,especially phagocytes (chemotaxis); (3) tagging of foreign organisms forphagocytosis (opsonization); and (4) destruction of invading organismsby the membrane attack complex (MAC attack). The central molecule is theC3 protein. It is an enzyme that is split into two fragments bycomponents of either the classical pathway or the alternative pathway.Antibodies, especially IgG and IgM, induce the classical pathway whilethe alternative pathway is nonspecifically stimulated by bacterialproducts like lipopolysaccharide (LPS). Briefly, the products of the C3split include a small peptide C3a that is chemotactic for phagocyticimmune cells and results in local vasodilation by causing the release ofC5a fragment from C5. The other part of C3, C3b coats antigens on thesurface of foreign organisms and acts to opsonize the organism fordestruction. C3b also reacts with other components of the complementsystem to form an MAC consisting of C5b, C6, C7, C8 and C9.

In general, IgG1 and IgG3 most effectively fix complement, IgG2 is lesseffective, and IgG4 does not activate complement. Complement activationis initiated by binding of Clq, a subunit of the first component C1 inthe cascade, to an antigen-antibody complex. Even though the bindingsite for Clq is located in the CH2 domain of the antibody, the hingeregion influences the ability of the antibody to activate the cascade.For example, recombinant immunoglobulins lacking a hinge region arereportedly unable to activate complement. Shin et al., 1992. Without theflexibility conferred by the hinge region, the Fab portion of theantibody bound to the antigen may not be able to adopt the conformationrequired to permit Clq to bind to CH2. See id. Hinge length andsegmental flexibility have been reported to correlate with complementactivation; however, the correlation is not absolute. Human IgG3molecules with altered hinge regions that are as rigid as IgG4, forexample, can still effectively activate the cascade.

The absence of a hinge region, or a lack of a functional hinge region,can also affect the ability of certain human IgG immunoglobulins to bindFc receptors on immune effector cells. Binding of an immunoglobulin toan Fc receptor facilitates antibody-dependent cell-mediatedcytotoxicity, which as noted above is presumed to be an importantmechanism for the elimination of tumor cells. The human IgG Fc receptor(FcR) family is divided into three groups, FcγRI (CD64), which iscapable of binding IgG with high affinity, and FcγRII (CD32) and FcγRIII(CD16), both of which are lower affinity receptors. The molecularinteraction between each of the three receptors and an immunoglobulinhas not been defined precisely, but experimental evidence indicates thatresidues in the hinge proximal region of the CH2 domain may be importantto the specificity of the interaction between the antibody and the Fcreceptor. IgG1 myeloma proteins and recombinant IgG3 chimeric antibodiesthat lack a hinge region are reportedly unable to bind FcγRI, perhapsbecause accessibility to CH2 is decreased. Shin et al., 1993 Intern.Rev. Immunol. 10:177, 178-79.

Unusual and apparently evolutionarily unrelated exceptions to the H₂L₂structure of conventional antibodies occur in some isotypes of theimmunoglobulins found in camelids (camels, dromedaries and llamas;Hamers-Casterman et al., 1993 Nature 363:446; Nguyen et al., 1998 J.Mol. Biol. 275:413), nurse sharks (Roux et al., 1998 Proc. Nat. Acad.Sci. USA 95:11804), and in the spotted ratfish (Nguyen, et al.,“Heavy-chain antibodies in Camelidae; a case of evolutionaryinnovation,” 2002 Immunogenetics 54(1):39-47). These antibodies canapparently form antigen-binding regions using only heavy chain variableregion, i.e., these functional antibodies are homodimers of heavy chainsonly (referred to as “heavy-chain antibodies” or “HCAbs”). In bothspecies, these variable regions often contain an extended thirdcomplementarity determining region (CDR3) that may help compensate forthe lack of a light chain variable region, and there are frequentdisulfide bonds between CDR regions that presumably help to stabilizethe binding site. Muyldermans et al., 1994 Prot. Engineer. 7:1129; Rouxet al., 1998. However, the precise function of the heavy chain-only“antibodies” is unknown, and the evolutionary pressure leading to theirformation has not been identified. See, e.g., Nguyen, et al., 2002,supra. Camelids, including camels, llamas, and alpacas, also expressconventional H₂L₂ antibodies, and the heavy chain-only antibodies thusdo not appear to be present in these animals simply as an alternativeantibody structure.

Variable regions (V_(H)H) of the camelid heavy chain-onlyimmunoglobulins and conventional (H₂L₂) heavy chain variable regionscontain amino acid differences, including differences at severalpositions that may be involved in the interface between conventionalV_(H) and V_(L) domains. Nguyen et al., 1998 J. Mol. Biol. 275:413;Muyldermans et al., 1994 Prot. Engineer. 7:1129. Camelid V_(H)Hreportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain. Hamers-Casterman etal., 1993 Nature 363:446. Interestingly, V_(H)H are encoded by achromosomal locus distinct from the V_(H) locus (Nguyen et al., 1998,supra), indicating that camelid B cells have evolved complex mechanismsof antigen recognition and differentiation. Thus, for example, llamaIgG1 is a conventional (H₂L₂) antibody isotype in which V_(H) recombineswith a constant region that contains hinge, CH1, CH2 and CH3 domains,whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that lackCH1 domains and that contain no light chains.

The classes of immunoglobulins have different physical and chemicalcharacteristics and they exhibit unique biological properties. Theirsynthesis occurs at different stages and rates during an immune responseand/or during the course of an infection. Their importance and functionsin host resistance (immunity) are different.

Immunoglobulin G (IgG), a protein with a molecular weight of about150,000 daltons (150 kD), is the predominant Ig in the serum. It makesup about 80% of the total antibody found in an animal at any given time,being 75% of the total serum antibody. It can diffuse out of the bloodstream into the extravascular spaces and it is the most common Ig foundthere. Its concentration in tissue fluids is increased duringinflammation, and it is particularly effective at the neutralization ofbacterial extracellular toxins and viruses. It also has opsonizingability and complement-fixing ability. The polypeptide composition ofthe Fc region of all IgG1 antibody molecules is relatively constantregardless of antibody specificity; however, as noted above, the Fabregions always differ in their exact amino acid sequences depending upontheir antigenic specificity. Specific amino acid regions of the Fcportion of the molecule are recognized by receptors on phagocytes andcertain other cells, and the Fc domain contains a peptide region thatwill bind to and activate complement, which is often required for themanifestation of antibody-mediated immunity. Because the IgG molecule isdivalent, it can cross-link antigen molecules, which may lead toprecipitation or agglutination of antigens; if IgG is bound to antigenon a microbial cell or surface, its Fc region may provide an extrinsicligand that will be recognized by specific receptors on phagocytes.Microbial cells or viruses coated with IgG molecules are opsonized forphagocytosis, and opsonized pathogens are taken up and destroyed muchmore readily by phagocytes than their non-opsonized counterparts. IgG,as well as IgM and IgA, will neutralize the activity of toxins,including bacterial exotoxins. Furthermore, cross-linked IgG moleculeson the surface of a cell can bind and activate complement from the serumand set off a cascade of reactions that can lead to destruction of thecell.

IgM is the first immunoglobulin to appear in the blood stream during thecourse of an infection. It is mainly confined to the bloodstream andprovides protection against blood-borne pathogens. IgM makes up about10% serum immunoglobulins, and is arranged to resemble a pentamer offive immunoglobulin molecules (having a molecular weight of about 900kD) tethered together at by their Fc domains. In addition to covalentlinkages between the monomeric subunits, the pentamer is stabilized by a15 kd polypeptide called J chain. IgM, therefore, has a theoretical“valence” of ten (i.e., it has ten exposed Fab domains). Probably, themost important role of IgM is its ability to function early in theimmune responses against blood-borne pathogens given its efficiency inagglutinating particulate antigens. IgM binds also complement stronglyand IgM antibodies bound to a microbial surface act as opsonins,rendering the microbe more susceptible to phagocytosis. In the presenceof complement and IgM whole microbial cells may be killed and lysed. Asnoted above, IgM also appears on the surfaces of mature B cells as atransmembranous monomer where it functions as an antigen receptor,capable of activating B cells when bound to antigen.

Gene rearrangement at the immunoglobulin loci during lymphoiddevelopment generates a repertoire of B lymphocytes that express adiversity of antigen receptors. The gene rearrangement, which iscatalysed by the rearrangement-activating gene (“RAG”) recombinase,integrates the immunoglobulin V, D and J gene segments to yieldproductively rearranged immunoglobulin genes that encode the heavy andlight chains of IgM antibodies. The diversity of IgM antibodies in thisprimary repertoire is achieved through combinatorial mechanisms (thechoice of V, D and J gene segments utilized in a particular antibody),as well as junctional diversity. The joining of V, D and J gene segmentsis somewhat imprecise, and nucleotides may be inserted at the junctionin a non-templated manner. There is therefore a very high degree ofresultant diversity at the V-D-J borders. This contributes in a majorway to the structural diversity of the third complementarity determiningregion of the antibody, a region that often plays a critical role inantigen recognition. This primary repertoire of IgM antibodies comprisesa few million different structures. The size of this repertoire meansthat any incoming antigen is likely to encounter an antibody thatrecognizes it with acceptable affinity. A high-affinity binding site isunlikely to be available for most incoming antigens (the repertoire isnot large enough), but the affinity of the available IgM antibodies inthe primary repertoire will vary from antigen to antigen. If an epitopeis re-iterated at high density on the surface of the antigen (e.g., arepeated structure on the surface of a virus or bacterium), then an IgMantibody may nevertheless be effective in mediating clearance of theorganism, despite the low affinity of the individual interaction betweenantigenic epitope and immunoglobulin combining site. The density of theepitopes may allow multivalent interactions with IgM, leading to ahigh-avidity interaction, providing that a suitable spacing of antigenicepitopes can occur. Nevertheless, to ensure an effective and specificresponse, especially when the concentration of antigen is low (as mayoccur when the body is faced with a very small number of infecting viralparticles), it would be preferable if high-affinity antibodies wereavailable for neutralizing, for example, an infecting organism. The sizeof the primary repertoire militates against the likelihood of suchhigh-affinity antibodies being present in this repertoire. The immunesystem therefore operates using a two-stage strategy. The primaryrepertoire of IgM antibodies is generated by a process of generearrangement and takes place prior to antigen encounter during earlylymphocyte development. However, once foreign antigen has beenencountered, those B cells in the primary repertoire that encodesuitable (albeit low-affinity) antibodies are selectively expanded andsubjected to an iterative alternation of directed hypermutation andantigen-mediated selection. This allows a significant maturation inaffinity of the antigen-specific antibodies that are produced. Antigentriggering also drives isotype switch recombination. Thus, in theabsence of external antigen stimulation and any maternally derivedimmunoglobulin, the serum will only contain a diversity of unmutated IgMmolecules that have been generated by gene rearrangement. Thisrepertoire shifts with age as a result of continuous antigen exposure,such that the majority of the serum immunoglobulin in older animals iscomposed of mutated IgG (and IgA) molecules whose specificities havedeveloped as a consequence of antigen selection.

IgA exists as a H₂L₂ monomer of about 160 kD in serum and, insecretions, as a dimer of the H₂L₂ monomer of about 400 kD. As with IgM,polymerization (dimerization) is via a J-chain. IgA has two subclassesbased on different heavy chains, IgA1 and IgA2. IgA1 is produced in bonemarrow and makes up most of the serum IgA. Both IgA1 and IgA2 aresynthesized in GALT (gut associated lymphoid tissues) to be secretedonto the mucosal surfaces. Because IgA may be synthesized locally andsecreted in the seromucous secretions of the body, it is sometimesreferred to as secretory antibody or sIgA. Quantitatively, IgA issynthesized in amounts greater than IgG, but it has a short half life inserum (6 days), and it is lost in secretory products. The concentrationof IgA in serum is about 15% of the total antibody. Secretion of dimericIgA is mediated by a 100 kD glycoprotein called secretory component. Itis the addition of the secretory piece to IgA molecules that accountsfor their ability to exit the body to mucosal surfaces via the exocrineglands. IgM can be transported similarly and makes up a small proportionof secretory antibodies. Secretory IgA is the predominant immunoglobulinpresent in gastrointestinal fluids, nasal secretions, saliva, tears andother mucous secretions of the body. IgA antibodies are important inresistance to infection of the mucosal surfaces of the body,particularly the respiratory, intestinal and urogenital tracts. IgA actsas a protective coating for the mucous surfaces against microbialadherence or initial colonization. It can also neutralize toxin activityon mucosal surfaces. Fc receptors for IgA-coated microorganisms found onmonocytes and neutrophils derived from the respiratory mucosa suggestthat IgA may have a role in the lung, at least, in opsonization ofpathogens. Secretory IgA is also transferred via the milk, i.e., thecolostrum, from a nursing mother to a newborn, which provides passiveimmunity to many pathogens, especially those that enter by way of the GItract.

IgE is an immunoglobulin of about 190 kD that accounts for about 0.002%of the total serum immunoglobulins. It is produced by plasma cells belowthe respiratory and intestinal epithelia. The majority of IgE is boundto tissue cells, especially mast cells. If an infectious agent succeedsin penetrating the IgA barrier, it comes up against the next line ofdefense, the MALT (mucosa-associated lymphoid tissues) system that ismanaged by IgE. IgE is bound very firmly to specific IgE Fc receptors onmast cells. Contact with antigen leads to release of mediators ofinflammation from the mast cells, which effectively recruits variousagents of the immune response including complement, chemotactic factorsfor phagocytes, T cells, etc. Although a well-known manifestation ofthis reaction is a type of immediate hypersensitivity reaction calledatopic allergy (e.g., hives, asthma, hay fever, etc.), the MALTresponses act as a defense mechanism because they amplify theinflammatory response and may facilitate rejection of a pathogen.

IgD is a molecule of about 175 kd that resembles IgG in its monomericform. IgD antibodies are found for the most part on the surfaces of Blymphocytes. The same cells may also carry IgM antibody. As noted above,it is thought that IgD and IgM function as mutually interacting antigenreceptors for control of B cell activation and suppression. Hence, IgDmay have an immunoregulatory function.

In addition to opsonization, activation of complement, and ADCC,antibodies have other functions in host defense including sterichindrance, toxin neutralization, agglutination, and precipitation. Withregard to steric hindrance, it is understood that antibodies combinewith the surfaces of microorganisms and may block or prevent theirattachment to susceptible cells or mucosal surfaces. Antibody against aviral component can block attachment of the virus to susceptible hostcells and thereby reduce infectivity. Secretory IgA can block attachmentof pathogens to mucosal surfaces. Toxin-neutralizing antibodies(antitoxins) can also react with a soluble bacterial toxin and block theinteraction of the toxin with its specific target cell or substrate.Antibodies can also combine with the surfaces of microorganisms orsoluble antigens and cause them to agglutinate or precipitate. Thisreduces the number of separate infectious units and makes them morereadily phagocytosed because the clump of particles is larger in size.Phagocytes may remove floccules or aggregates of neutralized toxin.

Antibodies have been proposed for use in therapy. Animals, includinghumans and mice have the ability to make antibodies able to recognize(by binding to) virtually any antigenic determinant and to discriminatebetween similar epitopes. Not only does this provide the basis forprotection against disease organisms, but it also makes antibodiesattractive candidates to target other types of molecules found in thebody, such as receptors or other proteins present on the surface ofnormal cells and molecules present uniquely on the surface of cancercells. Thus the remarkable specificity of antibodies makes thempromising agents for human therapy.

Initial antibody preparations available for use, such as intravenousgammaglobulins, included animal and human antisera that were used invivo to destroy bacteria (tetanus, pneumococcus) and neutralize virus(hepatitis A and B, rabies, cytomegalovirus, and varicella zoster) inthe blood of infected individuals. Possibly the most important earlyapplication was the use of endotoxins. However, there are problemsassociated with the use of antibodies in human therapy because theresponse of the immune system to any antigen, even the simplest, is“polyclonal,” i.e., the system manufactures antibodies of a great rangeof structures both in their binding regions as well as in their effectorregions. Polyclonal antibody treatment was also associated with unwantedside effects. In addition to the polyclonal nature of these antibodypreparations, there was the risk of infection from contaminatingviruses. Serum sickness and anaphylaxis were also considered limitingfactors. Furthermore, even if one were to isolate a singleantibody-secreting cell, and place it in culture, it would die out aftera few generations because of the limited growth potential of all normalsomatic cells.

Until the late 1970s, polyclonal antibodies obtained from the bloodserum of immunized animals provided the only source of antibodies forresearch or treatment of disease. Isolation of specific antibodies wasessentially impossible until Kohler and Milstein discovered how to make“monoclonal antibodies” that would have a single specificity, that wouldall be alike due to manufacture by a single clone of plasma cells andthat could be grown indefinitely. This technique was described in a 1975publication (Nature 256:495-97), and Köhler and Milstein received the1984 Nobel Prize in Medicine for their work.

The first step in Kohler and Milstein's technique for production ofmonoclonal antibodies involves immunizing an experimental animal withthe antigen of interest. In most of their experiments, Kohler andMilstein injected a mouse with sheep red blood cells. The mouse's bodyinitiates an immune response and begins producing antibodies specific tothe antigen. The mouse's spleen is then removed and B cells producingthe antibody of interest are isolated. Tumor-producing cells that havebeen grown in culture are then fused with the B lymphocytes usingpolyethylene glycol in order to produce a “hybridoma.” Only hybridomasresulting from the fusion will survive. The spleen lymphocyte has alimited life span, so any B cells that did not fuse with a myeloma willdie in the culture. Additionally, those cells that lack theantibody-producing aspect of the B cell will not secrete the enzymeHGPRT, which is required for growth in thehypoxathine-aminopterinthymidine (HAT) medium. The HAT medium, on whichthe cells are grown, inhibits the pathway for nucleotide synthesis.Cells that produce HGPRT can bypass this pathway and continue to grow.By placing the fused cells in a HAT medium, true hybridomas can beisolated. The isolated hybridoma cells are then screened for specificityto the desired antigen. Because each hybridoma descends from one B cell,it makes copies of only one antibody. The hybridoma that produces theantibody of interest is grown in culture to produce large amounts ofmonoclonal antibodies, which are then isolated for further use. Thetechnique is called somatic cell hybridization, and the resultinghybridoma (selected for both immortality and production of the specificantibody of interest) may be cultured indefinitely, i.e., it is apotenially immortal cell line.

Monoclonal antibodies are now widely used as diagnostic and researchreagents. However, their introduction into human therapy has been muchslower. One principal difficulty is that mouse antibodies are “seen” bythe human immune system as foreign. The human patient mounts an immuneresponse against them, producing HAMA (“human anti-mouse antibodies”).These not only cause the therapeutic antibodies to be quickly eliminatedfrom the host, but also form immune complexes that cause damage to thekidneys.

Two approaches have been used in an attempt to reduce the problem ofHAMA. The first is the production of chimeric antibodies in which theantigen-binding part (variable regions) of a mouse monoclonal antibodyis fused to the effector part (constant region) of a human antibodyusing genetic engineering. In a second approach, rodent antibodies havebeen altered through a technique known as complementarity determiningregion (CDR) grafting or “humanization.” In this process, the antigenbinding sites, which are formed by three CDRs of the heavy chain andthree CDRs of the light chain, are excised from cells secreting rodentmAb and grafted into the DNA coding for the framework of the humanantibody. Because only the antigen-binding site CDRs, rather than theentire variable domain of the rodent antibody are transplanted, theresulting humanized antibody (a second generation or “hyperchimeric”antibody) is reportedly less immunogenic than a first generationchimeric antibody. This process has been further improved to includechanges referred to as “reshaping” (Verhoeyen, et al., “Reshaping humanantibodies: grafting an anti-lysozyme activity,” 1988 Science239:1534-1536; Riechmann, et al., “Reshaping human antibodies fortherapy,” 1988 Nature 332:323-337; Tempest, et al., “Reshaping humanmonoclonal antibody to inhibit respiratory syncitial virus infection invivo,” Bio/Technol 1991 9:266-271), “hyperchimerization” (Queen, et al.,“A humanized antibody that binds to the human interleukin 2 receptor,”1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., “Humanizedantibodies for antiviral therapy,” 1991 Proc Natl Acad Sci USA88:2869±2873; Co, et al., “Chimeric and humanized antibodies withspecificity for the CD33 antigen,” 1992 J Immunol 148:1149-1154), and“veneering” (Mark, et al., “Derivation of therapeutically activehumanized and veneered anti-CD18 antibodies. In: Metcalf B W, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeuticpotential. New York: Plenum Press, 1994:291-312).

In the reshaping process on the basis of homology, the rodent variableregion is compared with the consensus sequence of the protein sequencesubgroup to which it belongs. Similarly, the selected human constantregion accepting framework is compared with its family consensussequence. Gussowal, et al., “Humanization of monoclonal antibodies,”1991 Meth Enzymol 203:99-121. The sequence analyses identify residues,which may have undergone mutation during the affinity maturationprocedure and may therefore be idiosyncratic. Inclusion of the morecommon human residues is said to lessen immunogenicity problems byreplacing human acceptor idiosyncratic residues. Further, the reshapingprocess is said to allow comparison of human and rodent consensussequences to identify any systematic “species” differences. RSV19antibodies were humanized by employing this procedure. Taylor et al.,“Humanized monoclonal antibody to respiratory syncitial virus,” 1991Lancet 337:1411-1412; Tempest, et al., “Reshaping a human monoclonalantibody to inhibit human respiratory syncitial virus infection invivo,” 1991 Bio/Technol 9:266-271.

Hyperchimerization is an alternative method of identifying residuesoutside CDR regions that are likely to be involved in the reconstitutionof binding activity. In this method, the human sequences are comparedwith murine variable region sequences and the one with highest homologyis selected as the acceptor framework. As in the reshaping procedure,the “idiosyncratic” residues are replaced by more commonly occurringhuman residues. The non-CDR residues that may be interacting with theCDR sequences are identified. Finally, it is determined which one ofthese residues is to be included in the variable region framework.Humanized antibodies against CD33 antigen were reportedly developed bythis method. Co, et al., “Chimeric and humanized antibodies withspecificity for the CD33 antigen,” 1992 J Immunol 148:1149-154. See alsoCarter, et al., “Humanization of an anti-p185 HER2 antibody for humancancer therapy,” 1992 Proc Natl Acad Sci USA 89:4285-4289.

The displayed surface of the protein is the primary determinant of itsimmunogenicity. A humanized murine antibody can thus be made lessimmunogenic by replacing exposed residues that differ from thosecommonly found in human antibodies. This method of humanization isreferred to as “veneering.” Appropriate replacement of the outerresidues may have little or no impact on the inner domains orinterdomain framework. Veneering is a two-step process. In the firststep, the most homologous human variable regions are selected andcompared by each single residue to the corresponding mouse variableregions. In the second step, the residues present in the human homologuereplace the mouse framework residues, which differ from its humanhomologue. This replacement involves only those residues that are on thesurface and at least partially exposed.

Nevertheless, it took more than a quarter century of research formonoclonal antibody technology and genetic engineering methods to resultin the development of immunoglobulin molecules for treatment of humandiseases. Indeed, it was not until the past five years that monoclonalantibodies became an expanding class of therapeutics. See Glennie M Jand van de Winkel J G, Drug Discov Today 2003 Jun. 1; 8(11):503-10;Souriau C and Hudson P J, “Recombinant antibodies for cancer diagnosisand therapy,” 2003 Expert Opin Biol Ther. 3(2):305-18. See also PendleyC, et al., “Immunogenicity of therapeutic monoclonal antibodies,” 2003Curr Opin Mol. Ther. 5(2):172-9.

All the same, an average of less than one therapeutic antibody per yearhas been introduced to the market beginning in 1986, eleven years afterthe publication of monoclonal antibodies. Five murine monoclonalantibodies were introduced into human medicine over a ten year periodfrom 1986-1995, including “muromonab-CD3” (OrthoClone OKT3®), whichbinds to a molecule on the surface of T cells and was launched in 1986to prevent acute rejection of organ transplants; “edrecolomab”(Panorex®), launched in 1995 for treatment of colorectal cancer;“odulimomab” (Antilfa®), launched in 1997 for use in transplantrejection; and, “ibritumomab” (Zevalin® yiuxetan), launched in 2002 foruse in non-Hodgkin's lymphoma. Additionally, one monoclonal Fab,“abciximab” (ReoPro®), was launched in 1995. It inhibits the clumping ofplatelets by binding the receptors on their surface that normally arelinked by fibrinogen and may be helpful in preventing reclogging of thecoronary arteries in patients who have undergone angioplasty. Threechimeric monoclonal antibodies were also launched: “rituximab”(Rituxan®), in 1997, which binds to the CD20 molecule found on most Bcells and is used to treat B cell lymphomas; “basiliximab” (Simulect®),in 1998 for transplant rejection; and “infliximab” (Remicade®) whichbinds to tumor necrosis factor-alpha (TNF-a), in 1998 for treatment ofrheumatoid arthritis and Crohn's disease. Additionally, “abciximab”(ReoPro®), a 47.6 kD Fab fragment of the chimeric human-murinemonoclonal antibody 7E3 that binds to the glycoprotein (GP) IIb/IIIareceptor of human platelets, was launched in 1995 as an adjunct topercutaneous coronary intervention for the prevention of cardiacischemic complications in patients undergoing percutaneous coronaryintervention. Finally, seven “humanized” monoclonals were launched from1997-2003: “daclizumab” (Zenapax®) in 1997, which binds to part of theIL-2 receptor produced at the surface of activated T cells and is usedto prevent acute rejection of transplanted kidneys; “palivizumab”(Synagis®) in 1998 for RSV; “trastuzumab” (Herceptin®) in 1998, whichbinds HER-2, a growth factor receptor found on breast cancers cells;“gemtuzumab” (Mylotarg®) in 2000, which is a conjugate of a monoclonalantibody that binds CD33, a cell-surface molecule expressed by thecancerous cells in acute myelogenous leukemia (AML) but not found on thenormal stem cells needed to repopulate the bone marrow; and“alemtuzumab” (MabCampath®) in 2001, which binds to CD52, a moleculefound on white blood cells and has produced temporary remission ofchronic lymphocytic leukemia; “adalimumab” (Humira® (D2E7)), a humananti-TNF monoclonal containing human-derived heavy chain and light chainvariable regions and human IgG:κ constant regions was launched in 2002for the treatment of rheumatoid arthritis; and, “omalizumab” (Xolair®),which binds to IgE and prevents it from binding to mast cells wasapproved in 2003 for the treatment of adults and adolescents over 12years of age with moderate to severe persistent asthma who have apositive skin test or in vitro reactivity to a perennial aeroallergenand whose symptoms are inadequately controlled with inhaledcorticosteroids.

Thus, protein engineering has been applied in an effort to diminishproblems related to immunogenicity of administered recombinantimmunoglobulin polypeptides and to try to alter antibody effectorfunctions. However, problems remain. For example, the majority of cancerpatients treated with rituximab relapse, generally within about 6-12months, and fatal infusion reactions within 24 hours of rituximabinfusion have been reported. These fatal reactions followed an infusionreaction complex that included hypoxia, pulmonary infiltrates, acuterespiratory distress syndrome, myocardial infarction, ventricularfibrillation or cardiogenic shock. Acute renal failure requiringdialysis with instances of fatal outcome has also been reported in thesetting of tumor lysis syndrome following treatment with rituximab, ashave severe mucocutaneous reactions, some with fatal outcome.Additionally, high doses of rituximab are required for intravenousinjection because the molecule is large, approximately 150 kDa, anddiffusion is limited into the lymphoid tissues where many tumor cellsreside.

Trastuzumab administration can result in the development of ventriculardysfunction and congestive heart failure, and the incidence and severityof cardiac dysfunction has been reported to be particularly high inpatients who received trastuzumab in combination with anthracyclines andcyclophosphamide. Trastuzumab administration can also result in severehypersensitivity reactions (including anaphylaxis), infusion reactions,and pulmonary events.

Patients receiving daclizumab immunosuppressive therapy are at increasedrisk for developing lymphoproliferative disorders and opportunisticinfections, and it is not known whether daclizumab use will have along-term effect on the ability of the immune system to respond toantigens first encountered during daclizumab-induced immunosuppression.

Hepatotoxicity, including severe hepatic veno-occlusive disease (VOD),has also been reported in association with the use of gemtuzumab as asingle agent, as part of a combination chemotherapy regimen, and inpatients without a history of liver disease or hematopoietic stem-celltransplant (HSCT). Patients who receive gemtuzumab either before orafter HSCT, patients with underlying hepatic disease or abnormal liverfunction, and patients receiving gemtuzumab in combinations with otherchemotherapy may be at increased risk for developing severe VOD. Deathfrom liver failure and from VOD has been reported in patients whoreceived gemtuzumab, and it has been cautioned that even carefulmonitoring may not identify all patients at risk or prevent thecomplications of hepatotoxicity.

Hepatotoxicity was also reported in patients receiving alemtuzumab.Serious and, in some rare instances fatal, pancytopenia/marrowhypoplasia, autoimmune idiopathic thrombocytopenia, and autoimmunehemolytic anemia have occurred in patients receiving alemtuzumabtherapy. Alemtuzumab can also result in serious infusion reactions aswell as opportunistic infections.

In patients treated with adalimumab, serious infections and sepsis,including fatalities, have been reported, as has the exacerbation ofclinical symptoms and/or radiographic evidence of demyelinating disease,and patients treated with adalimumab in clinical trials had a higherincidence of lymphoma than the expected rate in the general population.

Serious adverse reactions in clinical studies with omalizumab haveincluded malignancies and anaphylaxis, in which the observed incidenceof malignancy among omalizumab-treated patients (0.5%) was numericallyhigher than among patients in control groups (0.2%).

Smaller immunoglobulin molecules have been constructed in an effort toovercome various problems associated with whole immunoglobulin therapy.Single chain immunoglobulin variable region fragment polypeptides(scFvs) are made of an immunoglobulin heavy chain variable domain joinedvia a short linker peptide to an immunoglobulin light chain variabledomain. Huston et al., 1988 Proc. Natl. Acad. Sci. USA, 85:5879-83. Ithas been suggested that the smaller size of scFv molecules may lead tomore rapid clearance from plasma and more effective penetration intotissues than whole immunoglobulins. See, e.g., Jain, 1990 Cancer Res.50:814s-819s. An anti-tumor scFv was reported to show more rapid tumorpenetration and more even distribution through the tumor mass than thecorresponding chimeric antibody. Yokota et al., Cancer Res. 52:3402-08(1992).

Despite advantages that scFv molecules may have with regard toserotherapy, drawbacks to this therapeutic approach also exist. Forexample, rapid clearance of scFv may prevent delivery of a minimumeffective dose to the target tissue. Additionally, manufacturingadequate amounts of scFv for administration to patients has beenchallenging due to difficulties in expression and isolation of scFv thatadversely affect yields. During expression, scFv molecules lackstability and often aggregate due to pairing of variable regions fromdifferent molecules. Furthermore, production levels of scFv molecules inmammalian expression systems are reportedly low, which may limit thepotential for efficient manufacturing of scFv molecules for therapy.Davis et al., 1990 J. Biol. Chem. 265:10410-18; Traunecker et al., 1991EMBO J. 10:3655-59. Strategies for means to improve production have beenexplored, and reportedly include the addition of glycosylation sites tovariable regions. See, e.g., U.S. Pat. No. 5,888,773; Jost et al., 1994J. Biol. Chem. 269:26267-73. Another disadvantage to the use of scFvsfor therapy is the lack of effector function. An scFv that lacks thecytolytic functions, ADCC, and complement dependent-cytotoxicity may beless effective or ineffective for treating disease. Even thoughdevelopment of scFv technology began over 12 years ago, there arecurrently no scFv products approved for therapy.

Alternatively, it has been proposed that fusion of an scFv to anothermolecule, such as a toxin, could take advantage of the specificantigen-binding activity and the small size of an scFv to deliver thetoxin to a target tissue. Chaudary et al., 1989 Nature 339:394; Batra etal., 1991 Mol. Cell. Biol. 11:2200. Conjugation or fusion of toxins toscFvs has thus been offered as an alternative strategy to providepotent, antigen-specific molecules, but dosing with such conjugates orchimeras can be limited by excessive and/or non-specific toxicity due tothe toxin moiety of such preparations. Toxic effects may includesupraphysiological elevation of liver enzymes and vascular leaksyndrome, and other undesired effects. In addition, immunotoxins arethemselves highly immunogenic upon administration to a host, and hostantibodies generated against the immunotoxin limit potential usefulnessfor repeated therapeutic treatments of an individual.

Fusion proteins in which immunoglobulin constant region polypeptidesequences are present and nonimmunoglobulin sequences are substitutedfor the antibody variable regions have also been investigated. Forexample, CD4, the T cell surface protein recognized by HIV, wasrecombinantly fused to an immunoglobulin Fc effector domain, and anIL-2-IgG1 fusion protein reportedly effected complement-mediated lysisof IL-2 receptor-bearing cells. See Sensel et al., Chem. Immunol.65:129-158 (1997). An extensive introduction as well as detailedinformation about all aspects of recombinant antibody technology can befound in the textbook “Recombinant Antibodies” (John Wiley & Sons, NY,1999). A comprehensive collection of detailed antibody engineering labProtocols can be found in R. Kontermann and S. Dübel (eds.), “TheAntibody Engineering Lab Manual” (Springer Verlag, Heidelberg/New York,2000).

Diseases and disorders thought to be amenable to some type ofimmunoglobulin therapy include cancer and immune system disorders.Cancer includes a broad range of diseases, affecting approximately onein four individuals worldwide. Rapid and unregulated proliferation ofmalignant cells is a hallmark of many types of cancer, includinghematological malignancies. Although patients with a hematologicmalignant condition have benefited from advances in cancer therapy inthe past two decades, Multani et al., 1998 J. Clin. Oncology16:3691-3710, and remission times have increased, most patients stillrelapse and succumb to their disease. Barriers to cure with cytotoxicdrugs include, for example, tumor cell resistance and the high toxicityof chemotherapy, which prevents optimal dosing in many patients.

Nevertheless, patients have been treated with immunotherapeutics thattarget malignant cells, i.e., to antigens expressed on tumor cells. Withregard to the selection of tumor cell surface antigens suitable for useas immunotherapy targets, it is preferable that such a target antigen isnot expressed by normal tissues, particularly where the preservation ofsuch tissue is important to host survival. In the case of hematologicmalignancy, malignant cells express many antigens that are not expressedon the surfaces of stem cells or other essential cells. Treatment of ahematologic malignant condition using a therapeutic regimen thatdepletes both normal and malignant cells of hematological origin hasbeen acceptable where regeneration of normal cells from progenitors canoccur after therapy has ended. Additionally, the target antigen isdesirably expressed on all or virtually all clonogenic populations oftumor cells, and it is best that expression persists despite selectivepressure from immunoglobulin therapy. Strategies that employ selectionof a cell surface idiotype (e.g., a particular idiotope) as a target fortherapy of B cell malignancy have been limited by the outgrowth of tumorcell variants with altered surface idiotype expression, even where theantigen exhibits a high degree of tumor selectivity. Meeker et al., 1985N. Engl. J. Med. 312:1658-65. The selected antigen should also trafficproperly after an immunoglobulin binds to it. Shedding orinternalization of a cell surface target antigen after an immunoglobulinbinds to the antigen may allow tumor cells to escape destruction, thuslimiting the effectiveness of serotherapy. Finally, binding of animmunoglobulin to cell surface target antigens that transmit ortransduce cellular activation signals may result in improved functionalresponses to immunotherapy in tumor cells, and can lead to growth arrestand/or apoptosis. While all of these properties are important, thetriggering of apoptosis after an immunoglobulin binds to the targetantigen may also be a critical factor in achieving successfulserotherapy.

Antigens that have been tested as targets for serotherapy of B and Tcell malignancies include Ig idiotype (Brown et al., 1989 Blood73:651-61), CD19 (Hekman et al., 1991 Cancer Immunol. Immunother.32:364-72), Vlasveld et al., 1995 Cancer Immunol. Immunother. 40:37-47),CD20 (Press et al., 1987 Blood 69: 584-91), Maloney et al., 1997 J.Clin. Oncol. 15:3266-74), CD21 (Scheinberg et al., 1990 J. Clin. Oncol.8:792-803), CD5 (Dillman et al., 1986 J. Biol. Respn. Mod. 5:394-410),and CD52 (CAMPATH) (Pawson et al., 1997 J. Clin. Oncol. 15:2667-72). Ofthese, greater benefit for therapy of B cell lymphomas has been obtainedusing molecules that target CD20. Other targets have been limited bybiological properties of the antigen. For example, surface idiotype canbe altered through somatic mutation, allowing tumor cell escape. CD5,CD21, and CD19 are rapidly internalized after monoclonal antibodybinding, allowing tumor cells to escape destruction unless monoclonalantibodies are conjugated with toxin molecules. CD22 is expressed ononly a subset of B cell lymphomas, thereby limiting its usefulness,while CD52 is expressed on both T cells and B cells and may thereforegenerate counterproductive immunosuppression by depletion.

Treatment of patients with low grade or follicular B cell lymphoma usinga chimeric CD20 monoclonal antibody has been reported to induce partialor complete responses in patients. McLaughlin et al., 1996 Blood 88:90a(abstract, suppl. 1); Maloney et al., 1997 Blood 90:2188-95. However, asnoted above, tumor relapse commonly occurs within six months to oneyear. Further improvements in serotherapy are needed to induce moredurable responses, for example, in low grade B cell lymphoma, and toallow effective treatment of high-grade lymphoma and other B celldiseases.

Another approach has been to target radioisotopes to B cell lymphomasusing monoclonal antibodies specific for CD20. While the effectivenessof therapy is reportedly increased, associated toxicity from the long invivo half-life of the radioactive antibody increases also, sometimesrequiring that the patient undergo stem cell rescue. Press et al., 1993N. Eng. J. Med. 329:1219-1224; Kaminski et al., 1993 N. Eng. J. Med.329:459-65. Monoclonal antibodies to CD20 have also been cleaved withproteases to yield F(ab′)₂ or Fab fragments prior to attachment ofradioisotope. This has been reported to improve penetration of theradioisotope conjugate into the tumor and to shorten the in vivohalf-life, thus reducing the toxicity to normal tissues. However, thesemolecules lack effector functions, including complement fixation and/orADCC.

CD20 was the first human B cell lineage-specific surface moleculeidentified by a monoclonal antibody. It is a non-glycosylated,hydrophobic 35 kDa B cell transmembrane phosphoprotein that has bothamino and carboxy ends situated in the cytoplasm. Einfeld et al., 1988EMBO J. 7:711-17. CD20 is expressed by all normal mature B cells, but isnot expressed by precursor B cells. Natural ligands for CD20 have notbeen identified, and the function of CD20 in B cell biology is stillincompletely understood.

Anti-CD20 monoclonal antibodies affect the viability and growth andgrowth of B cells. Clark et al., 1986 Proc. Natl. Acad. Sci. USA83:4494-98. Extensive cross-linking of CD20 can induce apoptosis in Blymphoma cell lines, Shan et al., 1998 Blood 91:1644-52, andcross-linking of CD20 on the cell surface has been reported to increasethe magnitude and enhance the kinetics of signal transduction, forexample, as detected by measuring tyrosine phosphorylation of cellularsubstrates. Deans et al., 1993 J. Immunol. 146:846-53. Therefore, inaddition to cellular depletion by complement and ADCC mechanisms,Fc-receptor binding by CD20 monoclonal antibodies in vivo may promoteapoptosis of malignant B cells by CD20 cross-linking, consistent withthe theory that effectiveness of CD20 therapy of human lymphoma in aSCID mouse model may be dependent upon Fc-receptor binding by the CD20monoclonal antibody. Funakoshi et al., 1996 J. Immunotherapy 19:93-101.The presence of multiple membrane spanning domains in the CD20polypeptide (Einfeld et al., 1988 EMBO J. 7:711-17; Stamenkovic et al.,1988 J. Exp. Med. 167:1975-80; Tedder et al., 1988 J. Immunol.141:4388-4394), prevent CD20 internalization after antibody binding, andthis was recognized as an important feature for therapy of B cellmalignancies when a murine CD20 monoclonal antibody, 1F5, was injectedinto patients with B cell lymphoma, resulting in significant depletionof malignant cells and partial clinical responses. Press et al., 1987Blood 69:584-91.

Because normal mature B cells also express CD20, normal B cells aredepleted by anti-CD20 antibody therapy. Reff, M. E. et al., 1994 Blood83:435-445. After treatment is completed, however, normal B cells can beregenerated from CD20 negative B cell precursors; therefore, patientstreated with anti-CD20 therapy do not experience significantimmunosuppression. Depletion of normal B cells may also be beneficial indiseases that involve inappropriate production of autoantibodies orother diseases where B cells may play a role. A chimeric monoclonalantibody specific for CD20, consisting of heavy and light chain variableregions of mouse origin fused to human IgG1 heavy chain and human kappalight chain constant regions, reportedly retained binding to CD20 andthe ability to mediate ADCC and to fix complement. Liu et al., 1987 J.Immunol. 139:3521-26. The mechanism of anti-tumor activity of rituximab,discussed above, is thought to be a combination of several activities,including ADCC, complement fixation, and triggering of signals thatpromote apoptosis in malignant B cells, although the large size ofrituximab prevents optimal diffusion of the molecule into lymphoidtissues that contain malignant B cells, thereby limiting theseanti-tumor activities. Autoimmune diseases include autoimmune thyroiddiseases, which include Graves' disease and Hashimoto's thyroiditis. Inthe United States alone, there are about 20 million people who have someform of autoimmune thyroid disease. Autoimmune thyroid disease resultsfrom the production of autoantibodies that either stimulate the thyroidto cause hyperthyroidism (Graves' disease) or destroy the thyroid tocause hypothyroidism (Hashimoto's thyroiditis). Stimulation of thethyroid is caused by autoantibodies that bind and activate the thyroidstimulating hormone (TSH) receptor. Destruction of the thyroid is causedby autoantibodies that react with other thyroid antigens. Currenttherapy for Graves' disease includes surgery, radioactive iodine, orantithyroid drug therapy. Radioactive iodine is widely used, sinceantithyroid medications have significant side effects and diseaserecurrence is high. Surgery is reserved for patients with large goitersor where there is a need for very rapid normalization of thyroidfunction. There are no therapies that target the production ofautoantibodies responsible for stimulating the TSH receptor. Currenttherapy for Hashimoto's thyroiditis is levothyroxine sodium, and therapyis usually lifelong because of the low likelihood of remission.Suppressive therapy has been shown to shrink goiters in Hashimoto'sthryoiditis, but no therapies that reduce autoantibody production totarget the disease mechanism are known.

Rheumatoid arthritis (RA) is a chronic disease characterized byinflamation of the joints, leading to swelling, pain, and loss offunction. RA affects an estimated 2.5 million people in the UnitedStates. RA is caused by a combination of events including an initialinfection or injury, an abnormal immune response, and genetic factors.While autoreactive T cells and B cells are present in RA, the detectionof high levels of antibodies that collect in the joints, calledrheumatoid factor, is used in the diagnosis of RA. Current therapy forRA includes many medications for managing pain and slowing theprogression of the disease. No therapy has been found that can cure thedisease. Medications include nonsteroidal antiinflamatory drugs(NSAIDS), and disease modifying antirheumatic drugs (DMARDS). NSAIDS areuseful in benign disease, but fail to prevent the progression to jointdestruction and debility in severe RA. Both NSAIDS and DMARDS areassociated with signficant side effects. Only one new DMARD,Leflunomide, has been approved in over 10 years. Leflunomide blocksproduction of autoantibodies, reduces inflamation, and slows progressionof RA. However, this drug also causes severe side effects includingnausea, diarrhea, hair loss, rash, and liver injury.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused byrecurrent injuries to blood vessels in multiple organs, including thekidney, skin, and joints. SLE is estimated to affect over 500,000 peoplein the United States. In patients with SLE, a faulty interaction betweenT cells and B cells results in the production of autoantibodies thatattack the cell nucleus. These include anti-double stranded DNA andanti-Sm antibodies. Autoantibodies that bind phospholipids are alsofound in about half of SLE patients, and are responsible for bloodvessel damage and low blood counts. Immune complexes accumulate thekidneys, blood vessels, and joints of SLE patients, where they causeinflamation and tissue damage. No treatment for SLE has been found tocure the disease. NSAIDS and DMARDS are used for therapy depending uponthe severity of the disease. Plasmapheresis with plasma exchange toremove autoantibodies can cause temporary improvement in SLE patients.There is general agreement that autoantibodies are responsible for SLE,so new therapies that deplete the B cell lineage, allowing the immunesystem to reset as new B cells are generated from precursors, wouldoffer hope for long lasting benefit in SLE patients.

Sjogren's syndrome is an autoimmune disease characterized by destructionof the body's moisture-producing glands. Sjogren's syndrome is one ofthe most prevelant autoimmune disorders, striking up to an estimated 4million people in the United States. About half of people stricken withSjogren's syndrome also have a connective tissue disease, such as RA,while the other half have primary Sjogren's syndrome with no otherconcurrent autoimmune disease. Autoantibodies, including anti-nuclearantibodies, rheumatoid factor, anti-fodrin, and anti-muscarinic receptorare often present in patients with Sjogren's syndrome. Conventionaltherapy includes corticosteroids, and additional more effectivetherapies would be of benefit.

Immune thrombocytopenic purpura (ITP) is caused by autoantibodies thatbind to blood platelets and cause their destruction. Drugs cause somecases of ITP, and others are associated with infection, pregnancy, orautoimmune disease such as SLE. About half of all cases are classifiedas “idiopathic”, meaning the cause is unknown. The treatment of ITP isdetermined by the severity of the symptoms. In some cases, no therapy isneeded although in most cases immunosuppressive drugs, includingcorticosteroids or intravenous infusions of immune globulin to deplete Tcells, are provided. Another treatment that usually results in anincreased number of platelets is removal of the spleen, the organ thatdestroys antibody-coated platelets. More potent immunosuppressive drugs,including cyclosporine, cyclophosphamide, or azathioprine are used forpatients with severe cases. Removal of autoantibodies by passage ofpatients' plasma over a Protein A column is used as a second linetreatment in patients with severe disease. Additional more effectivetherapies are desired.

Multiple sclerosis (MS) is also an autoimmune disease. It ischaracterized by inflamation of the central nervous system anddestruction of myelin, which insulates nerve cell fibers in the brain,spinal cord, and body. Although the cause of MS is unknown, it is widelybelieved that autoimmune T cells are primary contributors to thepathogenesis of the disease. However, high levels of antibodies arepresent in the cerebral spinal fluid of patients with MS, and sometheories predict that the B cell response leading to antibody productionis important for mediating the disease. No B cell depletion therapieshave been studies in patients with MS, and there is no cure for MS.Current therapy is corticosteroids, which can reduce the duration andseverity of attacks, but do not affect the course of MS over time. Newbiotechnology interferon (IFN) therapies for MS have recently beenapproved but additional more effectiver therapies are desired.

Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorderthat is characterized by weakness of the voluntary muscle groups. MGeffects about 40,000 people in the united states. MG is caused byautoantibodies that bind to acetylcholine receptors expressed atneuromuscular junctions. The autoantibodies reduce or blockacetylcholine receptors, preventing the transmission of signals fromnerves to muscles. There is no known cure for mg. Common treatmentsinclude immunosuppression with corticosteroids, cyclosporine,cyclophosphamide, or azathioprine. Surgical removal of the thymus isoften used to blunt the autoimmune response. Plasmapheresis, used toreduce autoantibody levels in the blood, is effective in mg, but isshort-lived because the production of autoantibodies continues.Plasmapheresis is usually reserved for severe muscle weakness prior tosurgery. New and effective therapies would be of benefit.

Psoriasis affects approximately five million people, and ischaracterized by autoimmune inflammation in the skin. Psoriasis is alsoassociated with arthritis in 30% (psoriatic arthritis). Many treatments,including steroids, uv light retenoids, vitamin d derivatives,cyclosporine, methotrexate have been used but it is also plain thatpsoriasis would benefit from new and effective therapies. Scleroderma isa chronic autoimmune disease of the connective tissue that is also knownas systemic sclerosis. Scleroderma is characterized by an overproductionof collagen, resulting in a thickening of the skin, and approximately300,000 people in the United States have scleroderma, which would alsobenefit from new and effective therapies.

There is a clear need for improved compositions and methods to treatmalignacies, including B cell malignancies and disorders includingautoimmune diseases, disorders, and conditions, as well as otherdiseases, disorders, and conditions. The compositions and methods of thepresent invention described and claimed herein provide such improvedcompositions and methods as well as other advantages.

SUMMARY OF THE INVENTION

It is an aspect of the present invention to provide a bindingdomain-immunoglobulin fusion protein, comprising a binding domainpolypeptide that is fused or otherwise connected to an immunoglobulinhinge or hinge-acting region polypeptide, which in turn is fused orotherwise connected to a region comprising one or more native orengineered constant regions from an immunoglobulin heavy chain, otherthan CH1, for example, the CH2 and CH3 regions of IgG and IgA, or theCH3 and CH4 regions of IgE. The binding domain-immunoglobulin fusionprotein further comprises a region that comprises, consists essentiallyof, or consists of, a native or engineered immunoglobulin heavy chainCH2 constant region polypeptide (or CH3 in the case of a constructderived in whole or in part from IgE) that is fused or otherwiseconnected to the hinge region polypeptide and a native or engineeredimmunoglobulin heavy chain CH3 constant region polypeptide (or CH4 inthe case of a construct derived in whole or in part from IgE) that isfused or otherwise connected to the CH2 constant region polypeptide (orCH3 in the case of a construct derived in whole or in part from IgE).Such binding domain-immunoglobulin fusion proteins are capable of atleast one immunological activity selected from the group consisting ofantibody dependent cell-mediated cytotoxicity and complement fixation.Such binding domain polypeptides are also capable of binding orspecifically binding to a target, for example, a target antigen.

In certain embodiments, for example, the binding domain polypeptidecomprises at least one immunoglobulin variable region polypeptide thatis selected from a native or engineered immunoglobulin light chainvariable region polypeptide and/or a native or engineered immunoglobulinheavy chain variable region polypeptide. In certain further embodimentsthe binding domain-immunoglobulin fusion protein comprises a native orengineered immunoglobulin heavy chain variable region polypeptide,wherein the heavy chain variable region polypeptide is an engineeredhuman immunoglobulin heavy chain variable region polypeptide (or anengineered immunoglobulin heavy chain variable region polypeptide from anon-human species) comprising a mutation, substitution, or deletion ofan amino acid(s) at a location corresponding to any one or more of aminoacid positions 9, 10, 11, 12, 108, 110, and/or 112. Mutations,substitutions, or deletions of an amino acid(s) at a locationcorresponding to any one or more of amino acid positions 9, 10, 11, 12,108, 110, and/or 112 in a heavy chain variable region may be includedwithin a construct such as the construct corresponding to, for example,SEQ ID NO:212. In certain embodiments the immunoglobulin variable regionpolypeptide is derived from, for example, a human immunoglobulin, and incertain other embodiments the immunoglobulin variable region polypeptidecomprises a humanized immunoglobulin polypeptide sequence. In certainembodiments the immunoglobulin variable region polypeptide, whether ornot humanized, is derived from a murine immunoglobulin, or is derivedfrom an immunoglobulin from another species, including, for example arat, a pig, a monkey, or a camelid.

According to certain embodiments of the present invention, the bindingdomain polypeptide comprises, consists essentially of, or consists of,(a) at least one native or engineered immunoglobulin light chainvariable region polypeptide; (b) at least one native or engineeredimmunoglobulin heavy chain variable region polypeptide; and (c) at leastone linker polypeptide that is fused or otherwise connected to thepolypeptide of (a) and to the polypeptide of (b). In certain furtherembodiments the native or engineered immunoglobulin light chain variableregion and heavy chain variable region polypeptides are derived orconstructed from human immunoglobulins, and in certain other furtherembodiments the linker polypeptide comprises at least one polypeptideincluding or having as an amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQID NO:516]. In other embodiments the linker polypeptide comprises atleast two or three repeats of a polypeptide having as an amino acidsequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO:516]. In other embodiments thelinker comprises a glycosylation site, which in certain furtherembodiments is an asparagine-linked glycosylation site, an O-linkedglycosylation site, a C-mannosylation site, a glypiation site or aphosphoglycation site. In another embodiment at least one of a native orengineered immunoglobulin heavy chain CH2 constant region polypeptideand a native or engineered immunoglobulin heavy chain CH3 constantregion polypeptide is derived from an IgG or IgA human immunoglobulinheavy chain. In another embodiment at least one of a native orengineered immunoglobulin heavy chain CH3 constant region polypeptideand a native or engineered immunoglobulin heavy chain CH4 constantregion polypeptide is derived from an IgE human immunoglobulin heavychain.

An immunoglobulin hinge region polypeptide may comprise, consistessentially or, or consist of, for example, any of (1) any hinge orhinge-acting peptide or polypeptide that occurs naturally for example, ahuman immunoglobulin hinge region polypeptide including, for example, awild-type human IgG hinge or a portion thereof, a wild-type human IgAhinge or a portion thereof, a wild-type human IgD hinge or a portionthereof, or a wild-type human IgE hinge-acting region, i.e., IgE CH2, ora portion thereof, a wild-type camelid hinge region or a portion thereof(including a IgG1 llama hinge region or portion thereof, a IgG2 llamahinge region or portion thereof, and a IgG3 llama hinge region orportion thereof), a nurse shark hinge region or portion thereof, and/ora spotted ratfish hinge region or a portion thereof; (2) a mutated orotherwise altered or engineered hinge region polypeptide that containsno cysteine residues and that is derived or constructed from a wild-typeimmunoglobulin hinge region polypeptide having one or more cysteineresidues; (3) a mutated or otherwise altered or engineered hinge regionpolypeptide that contains one cysteine residue and that is derived froma wild-type immunoglobulin hinge region polypeptide having one or morecysteine residues; (4) a hinge region polypeptide that has been mutatedor otherwise altered or engineered to contain or add one or moreglycosylation sites, for example, an asparagine-linked glycosylationsite, an O-linked glycosylation site, a C-mannosylation site, aglypiation site or a phosphoglycation site; (5) a mutated or otherwisealtered or engineered hinge region polypeptide in which the number ofcysteine residues is reduced by amino acid substitution or deletion, forexample, a mutated or otherwise altered or engineered IgG1 or IgG4 hingeregion containing for example zero, one, or two cysteine residues, amutated or otherwise altered or engineered IgG2 hinge region containingfor example zero, one, two or three cysteine residues, a mutated orotherwise altered or engineered IgG3 hinge region containing for examplezero, one, two, three, or from four to ten cysteine residues, or amutated or otherwise altered or engineered human IgA1 or IgA2 hingeregion polypeptide that contains zero or only one or two cysteineresidues (e.g., an “SCC” hinge), a mutated or otherwise altered orengineered IgD hinge region containing no cysteine residues, or amutated or otherwise altered or engineered human IgE hinge-actingregion, i.e., IgE CH2 region polypeptide that contains zero or only one,two, three or four cysteine residues; or (6) any other connecting regionmolecule described or referenced herein or otherwise known or laterdiscovered as useful for connecting adjoining immunoglobulin domainssuch as, for example, a CH1 domain and a CH2 domain. For example, ahinge region polypeptide may be selected from the group consisting of(i) a wild-type human IgG1 immunoglobulin hinge region polypeptide, forexample, (ii) a mutated or otherwise altered or engineered human IgG1 orother immunoglobulin hinge region polypeptide that is derived orconstructed from a wild-type immunoglobulin hinge region polypeptidehaving three or more cysteine residues, wherein said mutated human IgG1or other immunoglobulin hinge region polypeptide contains two cysteineresidues and wherein a first cysteine of the wild-type hinge region isnot mutated, (iii) a mutated or otherwise altered or engineered humanIgG1 or other immunoglobulin hinge region polypeptide that is derivedfrom a wild-type immunoglobulin hinge region polypeptide having three ormore cysteine residues, wherein said mutated human IgG1 or otherimmunoglobulin hinge region polypeptide contains no more than onecysteine residue, and (iv) a mutated or otherwise altered or engineeredhuman IgG1 or other immunoglobulin hinge region polypeptide that isderived from a wild-type immunoglobulin hinge region polypeptide havingthree or more cysteine residues, wherein said mutated or otherwisealtered or engineered human IgG1 or other immunoglobulin hinge regionpolypeptide contains no cysteine residues. In certain embodiments, forexample, the immunoglobulin hinge region polypeptide is a mutated orotherwise altered or engineered hinge region polypeptide and exhibits areduced ability to dimerize, relative to a wild-type humanimmunoglobulin G or other wild type hinge region or hinge-actingpolypeptide.

The immunoglobulin heavy chain constant region polypeptides may be, forexample, native or engineered CH2 and CH3 domains of an isotype that ishuman IgG or human IgA. The immunoglobulin heavy chain constant regionpolypeptides may also be, for example, native or engineeredimmunoglobulin heavy chain constant region CH3 and CH4 polypeptides ofan isotype that is human IgE.

In certain other embodiments the target or target antigen may be, forexample, CD19 (B-lymphocyte antigen CD19, also referred to asB-lymphocyte surface antigen B4, or Leu-12), CD20 (B-lymphocyte antigen20, also referred to as B-lymphocyte surface antigen B1, Leu-16, orBp35), CD22 (B-cell receptor CD22, also referred to as Leu-14,B-lymphocyte cell adhesion molecule, or BL-CAM), CD37 (leukocyte antigenCD37), CD40 (B-cell surface antigen CD40, also referred to as TumorNecrosis Factor receptor superfamily member 5, CD40L receptor, or Bp50),CD80 (T lymphocyte activation antigen CD80, also referred to asActivation B7-1 antigen, B7, B7-1, or BB1), CD86 (T lymphocyteactivation antigen CD86, also referred to as Activation B7-2 antigen,B70, FUN-1, or BU63), CD137 (also referred to as Tumor Necrosis Factorreceptor superfamily member 9), CD152 (also referred to as cytotoxicT-lymphocyte protein 4 or CTLA-4), CD45 (Leukocyte common antigen, alsoreferred to as L-CA, T200, and EC 3.1.3.48), CD45RA (an isoform of CD45,and an antigen expressed on naïve or immature lymphocytes), CD45RB (anisoform of CD45), CD45RO (an isoform of CD45, and a common leukocyteantigen expressed on memory B and T cells), L6 (Tumor-associated antigenL6, also referred to as Transmembrane 4 superfamily member 1, Membranecomponent surface marker 1, or M3S1), CD2 (T-cell surface antigen CD2,also referred to as T-cell surface antigen T11/Leu-5, LFA-2, LFA-3receptor, Erythrocyte receptor, or Rosette receptor), CD28(T-cell-specific homodimer surface protein CD28, also referred to asTp44), CD30 (lymphocyte activation antigen CD30, also referred to asTumor Necrosis Factor receptor superfamily member 8, CD30L receptor, orKi-1), LD50 (also referred to as Intercellular adhesion molecule-3(ICAM3), or ICAM-R), CD54 (also referred to as Intercellular adhesionmolecule-1 (ICAM1), or Major group rhinovirus receptor), B7-H1 (ligandfor an immunoinhibitory receptor expressed by activated T cells, Bcells, and myeloid cells, also referred to as PD-L1; see Dong, et al.,“B7-H1, a third member of the B7 family, co-stimulates T-cellproliferation and interleukin-10 secretion,” 1999 Nat. Med.5:1365-1369), CD134 (also referred to as Tumor Necrosis Factor receptorsuperfamily member 4,OX40, OX40L receptor, ACT35 antigen, orTAX-transcriptionally activated glycoprotein 1 receptor), 41 BB (4-1 BBligand receptor, T-cell antigen 4-1BB, or T-cell antigen ILA), CD153(also referred to as Tumor Necrosis Factor ligand superfamily member 8,CD30 ligand, or CD30-L), CD154 (also referred to as Tumor NecrosisFactor ligand superfamily member 5, CD40 ligand, CD40-L, TNF-relatedactivation protein, TRAP, or T cell antigen Gp39), ICOS (InducibleCostimulator), CD3 (one or more of the delta, epsilon, gamma, eta and/orzeta chains, alone or in combination), CD4 (T-cell surface glycoproteinCD4, also referred to as T-cell surface antigen T4/Leu-3), CD25 (alsoreferred to as Interleukin-2 receptor alpha chain, IL-2 receptor alphasubunit, p55, or Tac antigen), CD8a (T-cell surface glycoprotein CD8alpha chain, also referred to as T-lymphocyte differentiation antigen,T8/Leu-2, and Lyt-2), CD11b (also referred to as Integrin alpha-M, Cellsurface glycoprotein MAC-1 alpha subunit, CR-3 alpha chain, Leukocyteadhesion receptor Mol, or Neutrophil adherence receptor), CD14 (Monocytedifferentiation antigen CD14, also referred to as Myeloid cell-specificleucine-rich glycoprotein or LPS receptor), CD56 (also referred to asNeural cell adhesion molecule 1), or CD69 (also referred to as EarlyT-cell activation antigen p60, Gp32/28, Leu-23, MLR-3, Activationinducer molecule, or AIM), and TNF factors (for example TNF-α). Theabove list of construct targets and/or target antigens is exemplary onlyand is not exhaustive.

In another aspect, the invention includes a binding construct (or apolynucleotide encoding such a construct) that comprises a CD154extracellular domain, or desired functional portion thereof. In oneembodiment of this aspect of the invention, for example, the bindingconstruct comprises a CD154 extracellular domain fused or otherwiseconnected to a second binding domain. The second binding domain, forexample, may comprise, consist essentially of, or consist of at leastone immunoglobulin variable region polypeptide. The at least oneimmunoglobulin variable region polypeptide may be a native or engineeredscFv. The native or engineered scFv may be a native or engineered scFvdisclosed or described herein. The second binding domain, including anative or engineered scFv, may be one that binds, for example, to any ofthe targets, including target antigens, disclosed or described herein,including but not limited to, for example, any of B7-H1, ICOS, L6, CD2,CD3, CD8, CD4, CD11b, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD37,CD40, CD45, CD50, CD54, CD56, CD69, CD80, CD86, CD134, CD137, CD152,CD153, or CD154.

In another embodiment the binding domain polypeptide comprises a CTLA-4extracellular domain, or desired functional portion thereof, and infurther embodiments at least one of the immunoglobulin heavy chainconstant region polypeptides selected from a CH2 constant regionpolypeptide and a CH3 constant region polypeptide is a human IgG1constant region polypeptide, either native or engineered.

In another further embodiment at least one of the immunoglobulin heavychain constant region polypeptides selected from a CH2 constant regionpolypeptide and a CH3 constant region polypeptide is a human IgAconstant region polypeptide, either native or engineered.

In another further embodiment at least one of the immunoglobulin heavychain constant region polypeptides selected from a CH3 constant regionpolypeptide and a CH4 constant region polypeptide is a human IgEconstant region polypeptide, either native or engineered.

Turning to another embodiment, the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyof, or consisting of, (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide; (b) anative or engineered immunoglobulin heavy chain CH2 (or IgE Ch3)constant region polypeptide that is fused or otherwise connected to thehinge region polypeptide; and (c) a native or engineered immunoglobulinheavy chain CH3 (or IgE CH4) constant region polypeptide that is fusedor otherwise connected to the CH2 (or IgE CH3) constant regionpolypeptide, wherein (1) the binding domain polypeptide comprises aCTLA-4 extracellular domain, or a portion thereof, that is capable ofbinding or specifically binding to at least one CTLA-4 ligand selectedfrom the group consisting of CD80 and CD86, (2) the immunoglobulin hingeregion polypeptide may be as described above or herein, and maycomprise, consist essentially of, or consist of, for example, apolypeptide that is selected from the group consisting of a native orengineered human IgA hinge region polypeptide, a native or engineeredhuman IgG1 hinge region polypeptide, and a native or engineered humanIgE CH2 region polypeptide (3) a immunoglobulin heavy chain constantregion polypeptide that comprises, consists essentially of, or consistsof, a polypeptide that is selected from the group consisting of a nativeor engineered human IgA heavy chain CH2 constant region polypeptide, anative or engineered human IgG1 heavy chain CH2 constant regionpolypeptide, and a native or engineered human IgE heavy chain CH3constant region polypeptide (4) a immunoglobulin heavy chain constantregion polypeptide that comprises, consists essentially of, or consistsof, a polypeptide that is selected from the group consisting of a nativeor engineered human IgA heavy chain CH3 constant region polypeptide, anative or engineered human IgG1 heavy chain CH3 constant regionpolypeptide, and a native or engineered human IgE heavy chain CH4constant region polypeptide, and (5) the binding domain-immunoglobulinfusion protein is capable of inducing at least one immunologicalactivity selected from the group consisting of antibody dependentcell-mediated cytotoxicity, CDC, and complement fixation. In a furtherembodiment, the binding domain-immunoglobulin fusion protein is capableof inducing two immunological activities selected from the groupconsisting of antibody dependent cell-mediated cytotoxicity, CDC, andcomplement fixation.

In another embodiment the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyof, or consisting of (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide may be as described above orherein, and may comprise, consist essentially of, or consist of, forexample, a native or engineered human IgE hinge-acting region, i.e., aIgE CH2 region polypeptide; (b) a first native or engineeredimmunoglobulin heavy chain constant region polypeptide that is fused orotherwise connected to the hinge region polypeptide, wherein said nativeor engineered constant region polypeptide comprises, consistsessentially of, or consists of, a native or engineered human IgE CH3constant region polypeptide; and (c) a second native or engineeredimmunoglobulin heavy chain constant region polypeptide that is fused orotherwise connected to the first native or engineered constant regionpolypeptide, wherein said native or engineered second constant regionpolypeptide comprises, consists essentially of, or consists of, a nativeor engineered human IgE CH4 constant region polypeptide and wherein (1)the binding domain-immunoglobulin fusion protein is capable of inducingat least one immunological activity selected from antibody dependentcell-mediated cytotoxicity and induction of an allergic responsemechanism, and (2) the binding domain polypeptide is capable of bindingor specifically binding to an antigen. In a further embodiment theantigen is a tumor antigen.

In certain other embodiments the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyof, or consisting of, (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide,wherein the binding domain polypeptide is capable of binding orspecifically binding to at least one antigen that is present on animmune effector cell and wherein the hinge region polypeptide may be asdescribed above or herein, and may comprise, consist essentially of, orconsist of, for example, a polypeptide selected from the groupconsisting of a native or engineered human IgA hinge region polypeptide,a native or engineered human IgG hinge region polypeptide, and a nativeor engineered human IgE hinge-acting region, i.e., IgE CH2 regionpolypeptide; (b) a first native or engineered immunoglobulin heavy chainconstant region polypeptide that is fused or otherwise connected to thehinge region polypeptide, wherein said first native or engineeredconstant region polypeptide comprises, consists essentially of, orconsists of, a polypeptide selected from the group consisting of anative or engineered human IgA CH2 constant region polypeptide, a nativeor engineered human IgG CH2 constant region polypeptide, and a native orengineered human IgE CH3 constant region polypeptide; (c) a secondnative or engineered immunoglobulin heavy chain constant regionpolypeptide that is fused or otherwise connected to the first constantregion polypeptide, wherein said second constant region polypeptidecomprises, consists essentially of, or consists of, a polypeptideselected from the group consisting of a native or engineered human IgACH3 constant region polypeptide, a native or engineered human IgG CH3constant region polypeptide, and a native or engineered human IgE CH4constant region polypeptide; and (d) a native or engineered plasmamembrane anchor domain polypeptide. In one example of this embodiment,the plasma membrane anchor domain polypeptide links to a membrane via anative or engineered glycosyl-phosphatidylinositol-linkage. In a furtherembodiment the plasma membrane anchor domain polypeptide comprises,consists essentially of, or consists of, a native or engineeredtransmembrane domain polypeptide. In another further embodiment themembrane anchor domain polypeptide comprises, consists essentially of,or consists of, a native or engineered transmembrane domain polypeptideand a native or engineered cytoplasmic tail polypeptide. In a stillfurther embodiment the cytoplasmic tail polypeptide comprises, consistsessentially of, or consists of, a native or engineered apoptosissignaling polypeptide sequence, which in a still further embodiment isderived or constructed from a native or engineered receptor death domainpolypeptide, a death domain, or a functional portion of either. In afurther embodiment the native or engineered death domain polypeptidecomprises, consists essentially of, or consists of, for example, anative or engineered polypeptide selected from an ITIM domain(immunoreceptor Tyr-based inhibition motif), an ITAM domain(immunoreceptor Tyr-based activation motif), TRAF, RIP, CRADD, FADD(Fas-associated death domain), TRADD (Tumor Necrosis Factor receptortype 1 associated DEATH domain protein), RAIDD (also referred to asRAID), CD95 (Tumor Necrosis Factor receptor superfamily member 6, alsoreferred to as FASL receptor, Apoptosis-mediating surface antigen FAS,FAS and Apo-1 antigen), TNFR1, and/or DR5 (death receptor-5). In anotherembodiment the native or engineered apoptosis signaling polypeptidesequence comprises, consists essentially of, or consists of, forexample, a polypeptide sequence derived from a native or engineeredcaspase polypeptide that is caspase-3 or caspase-8 or caspase-10,including caspase 8/FLICE/MACH/Mch5 and caspase 10/Flice2/Mch4. Inanother embodiment the plasma membrane anchor domain polypeptidecomprises, consists essentially of, or consists of, for example, anative or engineered glycosyl-phosphatidylinositol-linkage polypeptidesequence. In another embodiment the antigen that is present on an immuneeffector cell is, for example, CD2, CD16, CD28, CD30, CD32, CD40, CD50,CD54, CD64, CD80, CD86, B7-H1, CD134, CD137, CD152, CD153, CD154, ICOS,CD19, CD20, CD22, CD37, L6, CD3, CD4, CD25, CD8, CD11b, CD14, CD56, orCD69. In another embodiment the human IgG is a native or engineeredhuman IgG1. These binding domain-immunoglobulin fusion proteins may becapable of inducing, for example, at least one immunological activityselected from antibody dependent cell-mediated cytotoxicity and/orcomplement fixation and/or CDC, and are capable of binding orspecifically binding to a target, including, for example, a targetantigen. In other embodiments, the binding domain-immunoglobulin fusionproteins may be capable of inducing, for example, two immunologicalactivities selected from antibody dependent cell-mediated cytotoxicityand/or complement fixation and/or CDC, and are capable of binding orspecifically binding to a target. Immune effector cells include, forexample, granulocytes, mast cells, monocytes, macrophages, dendriticcells, neutrophils, eosinophils, basophils, NK cells, T cells (includingTh1 cells, Th2 cells, Tc cells, memory T cells, null cells, and largegranular lymphocytes, etc.), and B cells. This embodiment of theinvention further includes the use of such proteins for therapy, and,for example, the use of such vectors for in vivo and ex vivo genetherapy. The above lists of construct components and targets are notexhaustive and may include any desired target or component that mayfunction as, or be useful for the purposes, described herein.

In another embodiment, the invention provides a protein having a firstprotein motif that comprises, consists essentially of, or consists of,(1) a native or engineered immunoglobulin hinge region or hinge-actingregion (e.g., IgE CH2) polypeptide that is fused or otherwise connectedto (2) a native or engineered CH2 constant region polypeptide (or nativeor engineered IgE CH3 constant region polypeptide). Said first proteinmotif may be fused or otherwise connected to one or more other suchfirst protein motifs to form a second protein motif, the second proteinmotif being fused or otherwise connected to (3) a native or engineeredCH3 constant region (or a native or engineered IgE CH4 constant region)to form a third protein motif. Said first, second or third proteinmotifs may be fused or otherwise connected to one or more of theherein-described native or engineered plasma membrane anchor domainpolypeptides, including, for example, a native or engineeredtransmembrane domain polypeptide, and a native or engineeredtransmembrane domain polypeptide and a native or engineered cytoplasmictail polypeptide, such as for example, a native or engineered apoptosissignaling polypeptide sequence, which may be derived or constructed froma native or engineered receptor death domain polypeptide, a deathdomain, or a functional portion of either. Thus, a protein orpolynucleiotide within this aspect of the invention may be, for example,a Hinge-CH2-CH3-TransmembraneDomain-DeathDomain construct. It may alsobe, for example, a (Hinge-CH2)_(x)—CH3-TransmembraneDomain-DeathDomainconstruct, where X is from 2 to about 5, or such other number as may beneeded to achieve a desired length or Fc receptor binding and/orcomplement fixation function(s). This embodiment of the invention alsoincludes polynucleotides encoding such proteins, vectors including suchpolynucleotides, and host cells containing such polynucleotides andvectors. This embodiment of the invention further includes the use ofsuch proteins for therapy, and, for example, the use of suchpolynuceotides and/or vectors for in vivo and ex vivo gene therapy. Theinvention provides, in another embodiment, a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyof, or consisting of, (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide,wherein the binding domain polypeptide is capable of binding orspecifically binding to at least one antigen that is present on a cancercell surface and wherein the hinge region polypeptide may be asdescribed above or herein, and may comprise, consist essentially of, orconsist of, for example, a polypeptide selected from the groupconsisting of a native or engineered human IgA hinge region polypeptide,a native or engineered human IgG hinge region polypeptide, and a nativeor engineered human IgE hinge-acting region, i.e., IgE CH2, regionpolypeptide; (b) a first native or engineered immunoglobulin heavy chainconstant region polypeptide that is fused or otherwise connected to thehinge region polypeptide, wherein the first constant region polypeptidecomprises, consists essentially of, or consists of, a polypeptide thatis a native or engineered human IgA CH2 constant region polypeptide, anative or engineered human IgG CH2 constant region polypeptide, or anative or engineered human IgE CH3 constant region polypeptide; and (c)a second native or engineered immunoglobulin heavy chain constant regionpolypeptide that is fused or otherwise connected to the first constantregion polypeptide, wherein the second constant region polypeptidecomprises, consists essentially of, or consists of, a polypeptide thatis a native or engineered human IgA CH3 constant region polypeptide, anative or engineered human IgG CH3 constant region polypeptide, or anative or engineered human IgE CH4 constant region polypeptide. In afurther embodiment the human IgG polypepdides are native or engineeredhuman IgG1 polypeptides.

In another embodiment the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyof, or consisting of, (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide may be as described above orherein, and may comprises, consist essentially of, or consist of, forexample, a wild-type or engineered human IgA hinge region polypeptide;(b) a native or engineered immunoglobulin heavy chain CH2 constantregion polypeptide that is fused or otherwise connected to the hingeregion polypeptide, wherein said native or engineered CH2 constantregion polypeptide comprises, consists essentially of, or consists of, anative or engineered human IgA CH2 constant region polypeptide; and (c)a native or engineered immunoglobulin heavy chain CH3 constant regionpolypeptide that is fused or otherwise connected to the native orengineered CH2 constant region polypeptide, wherein the native orengineered CH3 constant region polypeptide comprises, consistsessentially of, or consists of, a polypeptide that is (i) a wild-typehuman IgA CH3 constant region polypeptide or other IgA region,preferably human or humanized, that is capable of associating with JChain, (ii) a mutated, altered or otherwise engineered human IgA CH3constant region polypeptide that is, for example, incapable ofassociating with a J chain, wherein (1) the bindingdomain-immunoglobulin fusion protein is capable of at least oneimmunological activity selected from the group consisting of antibodydependent cell-mediated cytotoxicity, CDC, and complement fixation, and(2) the binding domain polypeptide is capable of binding or specificallybinding to a target such as, for example, an antigen. In certain furtherembodiments the mutated human IgA CH3 constant region polypeptide thatis incapable of associating with a J chain is (i) a polypeptidecomprising, consisting essentially of, or consisting of, an amino acidsequence as set forth in SEQ ID NO:69 or (ii) a polypeptide comprising,consisting essentially of, or consisting of, an amino acid sequence asset forth in SEQ ID NO:74. In other embodiments, the IgA hinge regionpolypeptide is a native or engineered IgA1 hinge region polypeptide or anative or engineered IgA2 hinge region polypeptide. In still otherembodiments, the IgA hinge region polypeptide is different from awild-type IgA1 or IgA2 hinge region polypeptide by, for example, thealteration, substitution, or deltion of one or more of the cysteineresidues within said wild-type hinge region.

In certain other embodiments the present invention provides a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyof, or consisting of (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide; (b) anative or engineered immunoglobulin heavy chain CH2 constant regionpolypeptide that is fused or otherwise connected to the hinge regionpolypeptide, wherein the native or engineered CH2 constant regionpolypeptide comprises, consists essentially of, or consists of, a nativeor engineered llama CH2 constant region polypeptide that is a native orengineered llama IgG1 CH2 constant region polypeptide, a native orengineered llama IgG2 CH2 constant region polypeptide, or a native orengineered llama IgG3 CH2 constant region polypeptide; and (c) a nativeor engineered immunoglobulin heavy chain CH3 constant region polypeptidethat is fused or otherwise connected to the native or engineered CH2constant region polypeptide, wherein said native or engineered CH3constant region polypeptide comprises, consists essentially of, orconsists of, a native or engineered llama CH3 constant regionpolypeptide that is selected from the group consisting of a native orengineered llama IgG1 CH3 constant region polypeptide, a native orengineered llama IgG2 CH3 constant region polypeptide and a native orengineered llama IgG3 CH3 constant region polypeptide wherein (1) thebinding domain-immunoglobulin fusion protein is capable of at least oneimmunological activity selected from the group consisting of antibodydependent cell-mediated cytotoxicity, fixation of complement and CDC,and (2) the binding domain polypeptide is capable of binding orspecifically binding to a target, for example a target antigen. In afurther embodiment the immunoglobulin hinge region polypeptide, thenative or engineered llama CH2 constant region polypeptide and thenative or engineered llama CH3 constant region polypeptide comprisesequences derived from a native or engineered llama IgG1 polypeptide andthe fusion protein does not include a native or engineered llama IgG1CH1 domain. In certain embodiments the invention provides any of theabove described binding domain-immunoglobulin fusion proteins whereinthe hinge region polypeptide is mutated, engineered, or otherwisealtered to contain a glycosylation site, which in certain furtherembodiments is an asparagine-linked glycosylation site, an O-linkedglycosylation site, a C-mannosylation site, a glypiation site or aphosphoglycation site.

In certain embodiments the invention, there are provided any of theabove or herein described binding constructs, including bindingdomain-immunoglobulin fusion proteins, wherein a binding region orbinding domain polypeptide comprises two or more binding domainpolypeptide sequences wherein each of the binding domain polypeptidesequences is capable of binding or specifically binding to a target(s)such as an antigen(s), which target(s) or antigen(s) may be the same ormay be different. A native, for more preferably an engineered, IgD hingeis a desired connecting region between binding domains of a bispecificmolecule of the invention, i.e., one with two or more binding domains,preferably two. The wild type human IgD hinge has one cysteine thatforms a disulfide bond with the light chain in the native IgD structure.It is desirable to mutate or delete this cysteine in the human IgD hingefor use as a connecting region between binding domains of, for example,a bispecific molecule. Other amino acid changes or deletions oralterations in an IgD hinge that do not result in undesired hingeinflexibility are within the scope of the invention. Native orengineered IgD hinge regions from other species are also within thescope of the invention, as are humanized native or engineered IgD hingesfrom non-human species. The present invention also provides, in certainembodiments, a binding domain-immunoglobulin fusion protein, comprising,consisting essentially of, or consisting of (a) a binding domainpolypeptide that is fused or otherwise connected to an immunoglobulinhinge region polypeptide, wherein the hinge region polypeptide may be asdescribed above or herein, and may comprise, consist essentially of, orconsist of, for example, an alternative hinge region polypeptidesequence; (b) a first native or engineered immunoglobulin heavy chainconstant region, such as an IgG or IgA CH2 constant region polypeptide(or an IgE CH3 constant region polypeptide) that is fused or otherwiseconnected to the hinge region polypeptide; and (c) a second native orengineered immunoglobulin heavy chain constant region, such as an IgG orIgA CH3 constant region polypeptide (or an IgE CH4 constant regionpolypeptide) that is fused or otherwise connected to the first constantregion polypeptide, wherein: (1) the binding domain-immunoglobulinfusion protein is capable of at least one immunological activityselected from the group consisting of antibody dependent cell-mediatedcytotoxicity, CDC, and complement fixation, and (2) the binding domainpolypeptide is capable of binding or specifically binding to a target,such as an antigen.

Turning to another embodiment there is provided a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyof, or consisting of (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide,wherein the binding domain polypeptide is capable of binding orspecifically binding to at least one target, such as an antigen, that ispresent on a cancer cell surface and wherein the hinge regionpolypeptide may be as described above or herein, and may comprise,consist essentially of, or consist of, for example, an alternative hingeregion polypeptide sequence; (b) a first native or engineeredimmunoglobulin heavy chain constant region polypeptide that is fused orotherwise connected to the hinge region polypeptide, wherein said nativeor engineered constant region polypeptide comprises, consistsessentially of, or consists of, a polypeptide selected from the groupconsisting of a native or engineered human IgA CH2 constant regionpolypeptide, a native or engineered human IgG CH2 constant regionpolypeptide, and a native or engineered human IgE CH3 constant regionpolypeptide; and (c) a second immunoglobulin heavy chain constant regionpolypeptide that is fused or otherwise connected to the first constantregion polypeptide, wherein the second constant region polypeptidecomprises, consists essentially of, or consists of, a polypeptide thatis a native or engineered human IgA CH3 constant region polypeptide, anative or engineered human IgG CH3 constant region polypeptide, or anative or engineered human IgE CH4 constant region polypeptide. Incertain further embodiments the alternative hinge region polypeptidesequence comprises, consists essentially of, or consists of, apolypeptide sequence of at least ten continuous amino acids of an Ighinge region in any one of the constructs set out herein.

In certain embodiments the present invention provides polynucleotides orvectors (including cloning vectors and expression vectors) ortransformed or transfected cells, including isolated or purified or purepolynucleotides, vectors, and isolated transformed or transfected cells,encoding or containing any one of the above or herein describedpolypeptide or protein constructs of the invention, for example,including binding domain-immunoglobulin fusion proteins. Thus, invarious embodiments the invention provides a recombinant cloning orexpression construct comprising any such polynucleotide that is operablylinked to a promoter.

In other embodiments there is provided a host cell transformed ortransfected with, or otherwise containing, any such recombinant cloningor expression construct. Host cells include the cells of a subjectundergoing ex vivo cell therapy including, for example, ex vivo genetherapy.

In a related embodiment there is provided a method of producing apolypeptide or protein or other construct of the invention, for example,including a binding domain-immunoglobulin fusion protein, comprising thesteps of (a) culturing a host cell as described or provided for hereinunder conditions that permit expression of the construct, for example, abinding domain-immunoglobulin fusion protein; and (b) isolating theconstruct, for example, the binding domain-immunoglobulin fusion proteinfrom the host cell or host cell culture.

In another embodiment there is provided a pharmaceutical compositioncomprising any one of the above or herein described polypeptide orprotein or other constructs of the invention, for example (including,for example, binding domain-immunoglobulin fusion proteins), incombination with a physiologically acceptable carrier.

In another embodiment the invention provides a pharmaceuticalcomposition comprising, for example, an isolated, purified, or purepolynucleotide encoding any one of the polypeptide or protein constructsof the invention, for example (including, for example, bindingdomain-immunoglobulin fusion proteins), in combination with aphysiologically acceptable carrier, or for example, in combination with,or in, a gene therapy delivery vehicle or vector.

In another embodiment the invention provides a method of treating asubject having or suspected of having a malignant condition or a B celldisorder, comprising administering to a patient a therapeuticallyeffective amount of any of the pharmaceutical compositions described orclaimed herein.

In certain further embodiments the malignant condition or B celldisorder is a B cell lymphoma or B cell leukemia, or a diseasecharacterized by autoantibody production, and in certain other furtherembodiments the B cell disorder is, for example, rheumatoid arthritis,myasthenia gravis, Grave's disease, type I diabetes mellitus, multiplesclerosis or an autoimmune disease. In certain other embodiments themalignant condition is, for example, melanoma, myeloma, glioma,astrocytoma, lymphoma, leukemia, carcinoma, or sarcoma, and so on.

It is another aspect of the present invention to provide a bindingdomain-immunoglobulin fusion protein, comprising, consisting essentiallyor, or consisting of, (a) a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide is as described herein, and may beselected from the group consisting of (i) a mutated, engineered orotherwise altered hinge region polypeptide that contains no cysteineresidues and that is derived from a wild-type immunoglobulin hingeregion polypeptide having one or more cysteine residues, (ii) a mutated,engineered or otherwise altered hinge region polypeptide that containsone cysteine residue and that is derived from a wild-type immunoglobulinhinge region polypeptide having two or more cysteine residues, (iii) awild-type human IgA hinge region polypeptide, (iv) a mutated, engineeredor otherwise altered human IgA hinge region polypeptide that contains nocysteine residues, (v) a mutated, engineered or otherwise altered humanIgA hinge region polypeptide that contains one cysteine residue and (vi)a mutated, engineered or otherwise altered human IgA hinge regionpolypeptide that contains two cysteine residues; (b) a native orengineered immunoglobulin heavy chain CH2 constant region polypeptidethat is fused or otherwise connected to the hinge region polypeptide;and (c) a native or engineered immunoglobulin heavy chain CH3 constantregion polypeptide that is fused or otherwise connected to the CH2constant region polypeptide, wherein: (1) the bindingdomain-immunoglobulin fusion protein is capable of at least oneimmunological activity selected from the group consisting of antibodydependent cell-mediated cytotoxicity and complement fixation, and (2)the binding domain polypeptide is capable of binding or specificallybinding to an antigen. In one embodiment the immunoglobulin hinge regionpolypeptide is a mutated hinge region polypeptide, for example, and theresulting construct exhibits a reduced ability to dimerize, relative toa construct containing a wild-type human immunoglobulin G hinge regionpolypeptide. In another embodiment the binding domain polypeptidecomprises, consists essentially of, or consists of, at least one nativeor engineered immunoglobulin variable region polypeptide that is anative or engineered immunoglobulin light chain variable regionpolypeptide and/or a native or engineered immunoglobulin heavy chainvariable region polypeptide. In a further embodiment the native orengineered immunoglobulin variable region polypeptide is derived from ahuman immunoglobulin and, for example, may be humanized.

In another embodiment, the invention provides a bindingdomain-immunoglobulin fusion protein includes a binding domainpolypeptide that comprises, consists essentially of, or consists of, (a)at least one native or engineered immunoglobulin light chain variableregion polypeptide; (b) at least one native or engineered immunoglobulinheavy chain variable region polypeptide; and (c) at least one linkerpeptide that is fused or otherwise connected to the polypeptide of (a)and to the polypeptide of (b). In a further embodiment the native orengineered immunoglobulin light chain variable region and the native orengineered heavy chain variable region polypeptides are derived fromhuman immunoglobulins and may, for example, be humanized.

In another embodiment at least one of the native or engineeredimmunoglobulin heavy chain CH2 (or IgE CH3) constant region polypeptideand the native or engineered immunoglobulin heavy chain CH3 (or IgE CH4)constant region polypeptide is derived or constructed from a humanimmunoglobulin heavy chain. In another embodiment the native orengineered immunoglobulin heavy chain constant region CH2 and CH3polypeptides are of, or are derived or otherwise prepared or constructedfrom, an isotype selected from human IgG and human IgA. In anotherembodiment the target, for example, the target antigen is selected fromthe group consisting of CD16, CD19, CD20, CD37, CD40, CD45RO, CD80,CD86, CD137, CD152, and L6. In certain further embodiments of the abovedescribed fusion protein construct, the binding domain comprises,consists essentially of, or consists of, an scFv and the scFv contains alinker polypeptide that comprises, consists essentially of, or consistsof, at least one polypeptide comprising or having as an amino acidsequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO:516], and in certain otherembodiments the linker polypeptide comprises, consists essentially of,or consists of, at least three repeats of a polypeptide having as anamino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO:516]. In certainembodiments the immunoglobulin hinge region polypeptide comprises,consists essentially of, or consists of, a native or engineered humanIdG, IgA, IgD hinge region polypeptide, or a native or engineered IgECH2 region polypeptide. In certain embodiments the binding domainpolypeptide comprises, consists essentially of, or consists of, a nativeor engineered CD154 extracellular domain. In certain embodiments thebinding domain polypeptide comprises, consists essentially of, orconsists of, a native or engineered CD154 extracellular domain and atleast one a native or engineered immunoglobulin variable regionpolypeptide.

In other embodiments the invention provides an isolated polynucleotideencoding any of the constructs of the invention, for example, protein orpolypeptide constructs of the invention including bindingdomain-immunoglobulin fusion proteins, and in related embodiments theinvention provides a recombinant expression construct comprising such apolynucleotide, and in certain further embodiments the inventionprovides a host cell transformed or transfected with, or otherwisecontaining, such a recombinant expression construct. In anotherembodiment the invention provides a method of producing a construct ofthe invention, for example, a protein or polypeptide construct of theinvention such as a binding domain-immunoglobulin fusion protein,comprising the steps of (a) culturing a host cell that has beentransformed or transfected with, or otherwise made to contain, apolynucleotide construct of the invention under conditions that permitexpression of the construct, for example, a construct encoding a bindingdomain-immunoglobulin fusion protein; and (b) isolating the construct,for example, the binding domain-immunoglobulin fusion protein, from thehost cell culture.

The inventions described and claimed herein include novel moleculesuseful, for example, as therapeutics and other purposes includingdiagnostic and research purposes. Such molecules have, for example,antigen binding or other binding function(s) and one or more effectorfunctions. DNA constructs of the invention are useful in, for example,gene therapies, including in vivo and ex vivo gene therapies.

In one aspect, various constructs of the molecules of the inventioninclude molecules comprising a “binding region”, a “tail” region, and a“connecting” region that joins a binding region and a tail region.

Binding regions within the molecules of the invention may comprise, forexample, binding domains for desired targets, including antigen-bindingtargets. Binding domains for antigen-binding targets may comprise, forexample, single chain Fvs and scFv domains. In certain embodiments,molecules of the invention may comprise a binding region having at leastone immunoglobulin variable region polypeptide, which may be a lightchain or a heavy chain variable region polypeptide. In certainembodiments, molecules of the invention may comprise at least one suchlight chain V-region and one such heavy chain V-region and at least onelinker peptide that connects the V-regions. ScFvs useful in theinvention also include those with chimeric binding or other domains orsequences. Other ScFvs useful in the invention also include those withhumanized binding or other domains or sequences. In such embodiments,all or a portion of an immunoglobulin binding or other sequence that isderived from a non-human source may be “humanized” according torecognized procedures for generating humanized antibodies, i.e.,immunoglobulin sequences into which human Ig sequences are introduced toreduce the degree to which a human immune system would perceive suchproteins as foreign.

Example of scFvs useful in the invention, whether included as murine orother scFvs (including human scFvs), chimeric scFvs, or humanized scFvs,in whole or in part, include anti-human CD20 scFvs (for example, “2H7”scFvs), anti-human CD37 scFvs (for example, “G28-1” scFvs), anti-humanCD40 scFvs (for example, “G28-5” scFvs and “40.2.220” scFvs),anti-carcinoma-associated antigen scFvs (for example, “L6” scFvs),anti-CTLA-4 (CD152) scFvs (for example, “10A8” scFvs), anti-human CD28scFvs (for example, “2E12” scFvs), anti-murine CD3 scFvs (for example,“500A2” scFvs), anti-human CD3 scFvs (for example, G19-4 scFvs),anti-murine 4-1BB scFvs (for example, “1D8” scFvs), anti-human 4-1BBscFvs (for example, “5B9” scFvs), anti-human CD45RO (for example,“UCHL-1” scFvs), and anti-human CD16 (for example, “Fc2” scFvs).

scFvs useful in the invention also include scFvs, including chimeric andhumanized scFvs, having one or more amino acid substitutions. Apreferred amino acid substitution is at amino acid position 11 in thevariable heavy chain (the V_(H)). Such a substitution may be referred toherein as “XxxV_(H)11Zxx”. Thus, for example, where the normallyoccurring amino acid at position V_(H)11 is a Leucine, and a Serineamino acid residue is substituted therefore, the substitution isidentified as “L V_(H)11S” or “Leu V_(H)11Ser.” Other preferredembodiments of the invention include molecules containing scFvs whereinthe amino acid residue normally found at position V_(H)11 is deleted.Still other preferred embodiments of the invention include moleculescontaining scFvs wherein the amino acid residues normally found atpositions V_(H)10 and/or V_(H)11 and/or V_(H)12 are substituted ordeleted.

Other binding regions within the molecules of the invention may includedomains that comprise sites for glycosylation, for example, covalentattachment of carbohydrate moieties such as monosaccharides oroligosaccharides.

Still other binding regions within molecules of the invention includepolypeptides that may comprise proteins or portions thereof that retainthe ability to specifically bind another molecule, including an antigen.Thus, binding regions may comprise or be derived from hormones,cytokines, chemokines, and the like; cell surface or soluble receptorsfor such polypeptide ligands; lectins; intercellular adhesion receptorssuch as specific leukocyte integrins, selectins, immunoglobulin genesuperfamily members, intercellular adhesion molecules (ICAM-1, -2, -3)and the like; histocompatibility antigens; and so on. Binding regionsderived from such molecules generally will include thoss portions of themolecules necessary or desired for binding to a target.

Certain constructs include binding regions that comprise receptor orreceptor-binding domains. Receptor domains useful for binding to atarget include, for example, a CD154 extracellular domain, or a CTLA-4extracellular domain. In another example, the binding domain may includea first portion comprising, consisting essentially or, or consisting of,a CD154 extracellular domain and a second portion comprising, consistingessentially or, or consisting of, at least one immunoglobulin variableregion polypeptide, said second portion including, for example, an scFvor a V_(H). Examples of other cell surface receptors that may comprise,consist essentially or, or consist of, or a portion of which mayprovide, a binding region or binding domain polypeptide, include, forexample, HER1, HER2, HER3, HER4, epidermal growth factor receptor(EGFR), vascular endothelial cell growth factor, vascular endothelialcell growth factor receptor, insulin-like growth factor-I, insulin-likegrowth factor-II, transferrin receptor, estrogen receptor, progesteronereceptor, follicle stimulating hormone receptor (FSH-R), retinoic acidreceptor, MUC-1, NY-ESO-1, Melan-A/MART-1, tyrosinase, Gp-100, MAGE,BAGE, GAGE, any of the CTA class of receptors including in particularHOM-MEL-40 antigen encoded by the SSX2 gene, carcinoembyonic antigen(CEA), and PyLT. Additional cell surface receptors that may be sourcesof binding regions or binding domain polypeptides include, for example,CD2, 4-1BB, 4-1BB ligand, CD5, CD10, CD27, CD28, CD152/CTLA-4, CD40,interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-17 (IL-17) andinterleukin-17 receptor (IL-17R). Still other cell surface receptorsthat may be sources of binding regions and/or binding domainpolypeptides include, for example, CD59, CD48, CD58/LFA-3, CD72, CD70,CD80/B7.1, CD86/B7.2, B7-H1/B7-DC, IL-17, CD43, ICOS, CD3 (e.g., gammasubunit, epsilon subunit, delta subunit), CD4, CD25, CD8, CD11b, CD14,CD56, CD69 and VLA-4 (α₄β₇). The following cell surface receptors aretypically associated with B cells: CD19, CD20, CD22, CD30, CD153 (CD30ligand), CD37, LD50 (ICAM-3), CD106 (VCAM-1), CD54 (ICAM-1),interleukin-12, CD134 (OX40), CD137 (41BB), CD83, and DEC-205. Theselists are not exhaustive. Binding regions such as those set forth abovemay be connected, for example, by a native or engineered IgD hingeregion polypeptide, preferably a human or humanized native or engineeredIgD hinge region polypeptide. The invention thus further providesconstructs that comprise, consist essentially of, or consist of, twobinding regions, for example, an scFv and a cell surface receptor (orportion thereof), connected by a third molecule, for example, an IgDhinge region polypeptide as described herein.

Various molecules of the invention described and claimed herein includea connecting region joining one end of the molecule to another end. Suchconnecting regions may comprise, for example, immunoglobulin hingeregion polypeptides, including any hinge peptide or polypeptide thatoccurs naturally. A connecting region may also include, for example, anyartificial peptide or other molecule (including, for example,non-peptide molecules, partial peptide molecules, and peptidomimetics,etc.) useful for joining the tail region and the binding region. Thesemay include, for example, alterations of molecules situated in animmunoglobulin heavy chain polypeptide between the amino acid residuesresponsible for forming intrachain immunoglobulin-domain disulfide bondsin CH1 and CH2 regions. Naturally occurring hinge regions include thoselocated between the constant region domains, CH1 and CH2, of animmunoglobulin. Useful immunoglobulin hinge region polypeptides include,for example, human immunoglobulin hinge region polypeptides and llama orother camelid immunoglobulin hinge region polypeptides. Other usefulimmunoglobulin hinge region polypeptides include, for example, nurseshark and spotted ratfish immunoglobulin hinge region polypeptides.Human immunoglobulin hinge region polypeptides include, for example,wild type IgG hinges including wild-type human IgG1 hinges, humanIgG-derived immunoglobulin hinge region polypeptides, a portion of ahuman IgG hinge or IgG-derived immunoglobulin hinge region, wild-typehuman IgA hinge region polypeptides, human IgA-derived immunoglobulinhinge region polypeptides, a portion of a human IgA hinge regionpolypeptide or IgA-derived immunoglobulin hinge region polypeptide,wild-type human IgD hinge region polypeptides, human Ig-D derivedimmunoglobulin hinge region polypeptides, a portion of a human IgD hingeregion polypeptide or IgD-derived immunoglobulin hinge regionpolypeptide, wild-type human IgE hinge-acting region, i.e., IgE CH2region polypeptides (which generally have 5 cysteine residues), humanIgE-derived immunoglobulin hinge region polypeptides, a portion of ahuman IgE hinge-acting region, i.e., IgE CH2 region polypeptide orIgE-derived immunoglobulin hinge region polypeptide, and so on. Apolypeptide “derived from” or that is “a portion or fragment of” animmunoglobulin polypeptide chain region regarded as having hingefunction has one or more amino acids in peptide linkage, for example15-115 amino acids, preferably 95-110, 80-94, 60-80, or 5-65 aminoacids, preferably 10-50, more preferably 15-35, still more preferably18-32, still more preferably 20-30, still more preferably 21, 22, 23,24, 25, 26, 27, 28 or 29 amino acids. Llama immunoglobulin hinge regionpolypeptides include, for example, an IgG1 llama hinge. The connectingregion may comprise a stretch of consecutive amino acids from animmunoglobulin hinge region. For example, the connecting region cancomprise at least five consecutive hinge region amino acids, at leastten consecutive hinge region amino acids, at least fifteen consecutivehinge region amino acids, at least 20 consecutive hinge region aminoacids, and at least twenty five or more consecutive hinge region aminoacids from human IgG hinge, human IgA hinge, human IgE hinge, camelidhinge region, IgG1 llama hinge region, nurse shark hinge region, andspotted ratfish hinge region, including for example an IgG₁ hingeregion, a IgG₂ hinge region, a IgG₃ hinge region, an IgG₃ hinge region,and an IgG₄ hinge region.

Such connecting regions also include, for example, mutated or otherwisealtered or engineered immunoglobulin hinge region polypeptides. Amutated or otherwise altered or engineered immunoglobulin hinge regionpolypeptide may comprise, consist essentially of, or consist of, a hingeregion that has its origin in an immunoglobulin of a species, of animmunoglobulin isotype or class, or of an immunoglobulin subclass thatis the same or different from that of any included native or engineeredCH2 and CH3 domains. Mutated or otherwise altered or engineeredimmunoglobulin hinge region polypeptides include those derived orconstructed from, for example, a wild-type immunoglobulin hinge regionthat contains one or more cysteine residues, for example, a wild-typehuman IgG or IgA hinge region that naturally comprises three cysteines.In such polypeptides the number of cysteine residues may be reduced byamino acid substitution or deletion or truncation, for example. Thesepolypeptides include, for example, mutated human or other IgG1 or IgG4hinge region polypeptides containing zero, one, or two cysteineresidues, and mutated human or other IgA1 or IgA2 hinge regionpolypeptides that contain zero, one, or two cysteine residues. Mutatedor otherwise altered or engineered immunoglobulin hinge regionpolypeptides include those derived or constructed from, for example, awild-type immunoglobulin hinge region that contains three or morecysteine residues, for example, a wild-type human IgG2 hinge region(which has 4 cysteines) or IgG4 hinge region (which has 11 cysteines).Mutated or otherwise altered or engineered immunoglobulin hinge regionpolypeptides include those derived or constructed from, for example, anIgE CH2 wild-type immunoglobulin region that generally contains fivecysteine residues. In such polypeptides the number of cysteine residuesmay be reduced by one or more cysteine residues by amino acidsubstitution or deletion or truncation, for example. Also included arealtered hinge region polypeptides in which cysteine residues in thehinge region are substituted with serine or one or more other aminoacids that are less polar, less hydrophobic, more hydrophilic, and/orneutral. Such mutated immunoglobulin hinge region polypeptides include,for example, mutated hinge region polypeptides that contain one cysteineresidue and that are derived from a wild-type immunoglobulin hingeregion polypeptide having two or more cysteine residues, such as amutated human IgG or IgA hinge region polypeptide that contains onecysteine residue and that is derived from a wild-type human IgG or IgAregion polypeptide. Connecting region polypeptides includeimmunoglobulin hinge region polypeptides that are compromised in theirability to form interchain, homodimeric disulfide bonds.

Mutated immunoglobulin hinge region polypeptides also include mutatedhinge region polypeptides that exhibit a reduced ability to dimerize,relative to a wild-type human immunoglobulin G hinge region polypeptide,and mutated hinge region polypeptides that allow expression of a mixtureof monomeric and dimeric molecules. Mutated immunoglobulin hinge regionpolypeptides also include hinge region polypeptides engineered tocontain a glycosylation site. Glycosylation sites include, for example,an asparagine-linked glycosylation site, an O-linked glycosylation site,a C-mannosylation site, a glypiation site, and a phosphoglycation site.

Specific connecting regions useful in molecules of the inventiondescribed and claimed herein include, for example, the following 18amino acid sequences, DQEPKSCDKTHTCPPCPA (SEQ ID NO:10),DQEPKSSDKTHTSPPSPA (SEQ ID NO:18), and DLEPKSCDKTHTCPPCPA (SEQ IDNO:12). Other specific connecting regions include, for example, themutant hinges within the sequences referred to herein as “2H7 scFv(SSS-S)H WCH2 WCH3” and “2H7 scFv (CSS)H WCH2 WCH3”, and the humanIgA-derived hinge referred to herein as “2H7 scFv IgAH WCH2 WCH3”.

Tail regions within the molecules of the invention may include heavychain constant region immunoglobulin sequences. Tail regions may thusinclude, for example, a polypeptide having at least one of animmunoglobulin heavy chain CH2 constant region polypeptide and animmunoglobulin heavy chain CH3 constant region polypeptide. At least oneof the immunoglobulin heavy chain CH2 constant region polypeptide andthe immunoglobulin heavy chain CH3 constant region polypeptide may bederived from a human immunoglobulin heavy chain. Thus, for example, CH2and/or CH3 polypeptides may be derived from human IgG, human IgA, orhuman IgD molecules. Tail regions may also include, for example, apolypeptide having at least one of an immunoglobulin heavy chain CH3constant region polypeptide and an immunoglobulin heavy chain CH4constant region polypeptide. At least one of the immunoglobulin heavychain CH3 constant region polypeptide and the immunoglobulin heavy chainCH4 constant region polypeptide may be derived from a humanimmunoglobulin heavy chain. Thus, for example, CH3 and/or CH4polypeptides may be derived from human IgE. An immunoglobulin heavychain CH2 region polypeptide included within a molecule of the inventionmay, for example, be from the IgG1, IgG2, IgG3 and/or IgG4 subclasses.An immunoglobulin heavy chain CH3 region polypeptide included within amolecule of the invention may also, for example, be from the IgG1, IgG2,IgG3 and/or IgG4 subclasses. Additionally, both the immunoglobulin heavychain CH2 region polypeptide and the immunoglobulin heavy chain CH2region polypeptide included within a molecule of the invention may, forexample, be from the IgG1, IgG2, IgG3 and/or IgG4 subclasses. In othermolecules of the invention at least one of the immunoglobulin heavychain constant region polypeptides selected from a CH2 constant regionpolypeptide and a CH3 constant region polypeptide is a human IgAconstant region polypeptide. An immunoglobulin heavy chain CH2 regionpolypeptide included within a molecule of the invention may, forexample, be from the IgA1 and/or IgA2 subclasses. An immunoglobulinheavy chain CH3 region polypeptide included within a molecule of theinvention may also, for example, be from the IgA1 and/or IgA2subclasses. Additionally, both the immunoglobulin heavy chain CH2 regionpolypeptide and the immunoglobulin heavy chain CH2 region polypeptideincluded within a molecule of the invention may, for example, be fromthe IgA1 and/or IgA2 subclasses. In still other molecules of theinvention, the tail region may comprise or consist essentially of a CH2and/or CH3 constant region polypeptide comprising a polypeptide fromhuman IgA and/or human IgE. In other embodiments, for example, the tailregion within a molecule of the invention may include an immunoglobulinheavy chain CH2 and/or CH3 constant region polypeptide that is a mutated(for example, a mutated IgA CH3 constant region polypeptide that isincapable of associating with a J chain in which, for example, the IgACH3 constant region polypeptide is of human origin). The tail region mayalso comprise, consist essentially of, or consist of an extracellularportion of a protein from the TNF superfamily, for example, CD154.

For molecules of the invention intended for use in humans, these regionswill typically be substantially or completely human to minimizepotential human immune responses against the molecules and to provideappropriate effector functions. In certain embodiments of the invention,for example, the tail region includes a human IgG1 CH3 region sequence,a wild-type IgA heavy chain constant region polypeptide sequence that iscapable or incapable of associating with J chain.

In preferred embodiments of the invention, a CH1 domain is not includedin the tail region of the molecule, and the carboxyl end of the bindingregion is joined to the amino terminus of a CH2 portion of a tail regioneither directly or indirectly. A binding region may be indirectly joinedto a tail region, for example via a connecting region polypeptide orother connecting molecule.

The invention also includes molecules that have mutated CH2 and/or CH3sequences within a tail region. For example, a molecule of the inventionmay include a mutated Fc domain that has one or more mutationsintroduced into the CH2, CH3 and/or CH4 domains. In certain embodimentsof the invention, molecules may include an IgA CH3 constant regionpolypeptide such as a human IgA CH3 constant region polypeptide in whichtwo or more residues from the C-terminus have been deleted to yield atruncated CH3 constant region polypeptide. In other embodiments of theinvention, molecules include a mutated human IgA CH3 constant regionpolypeptide that is incapable of associating with a J chain thatcomprises a C-terminal deletion of either four or 18 amino acids.However, the invention need not be so limited, such that moleculescontaining the mutated IgA CH3 constant region polypeptide may comprisea deletion of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21-25, 26-30 or more amino acids, so long as the fusionprotein is capable of specifically binding an antigen and capable of atleast one immunological activity such as ADCC, CDC or complementfixation. The invention also includes molecules containing a tail regionthat comprises a mutated IgA CH3 constant region polypeptide that isincapable of associating with a J chain by virtue of replacement of thepenultimate cysteine, or by chemical modification of that amino acidresidue, in a manner that prevents interchain disulfide bond formation.

Various molecules of the invention include, for example, a bindingdomain scFv-fusion protein having a binding domain polypeptidecomprising, consisting essentially of, or consisting of, (a) at leastone immunoglobulin light chain variable region polypeptide, (b) at leastone immunoglobulin heavy chain variable region polypeptide, and at leastone linker peptide that joins the polypeptide of (a) and the polypeptideof (b). Such polypeptides may, for example, be derived from humanimmunoglobulins or non-human immunoglobulins.

Thus, in one aspect, the invention includes a non-naturally occurringsingle chain protein and/or V_(H) protein and/or V_(L) protein, or adesired portion of any of the above, including a first polypeptidecomprising a binding domain polypeptide capable of binding to a targetmolecule, a second polypeptide comprising a flexible or other desiredlinker attached to said first polypeptide, a third polypeptidecomprising a tail region, for example, an N-terminally truncatedimmunoglobulin heavy chain constant region polypeptide (or desiredportion thereof) attached to the second polypeptide. The flexible linkermay comprise, consist essentially of, or consist of, an immunoglobulinhinge region or portion thereof that has been mutated or otherwisealtered or engineered, for example, one that contains a number ofcysteine residues that is less than the number of cysteine residuespresent in the wild type immunoglobulin hinge region or portion (forexample, zero, one, or two cysteines in the case of IgG1 or IgG4), andwherein said non-naturally occurring single-chain protein is capable ofat least one immunological activity, for example, ADCC, CDC, and/orcomplement fixation. The single chain protein may be capable of twoimmunological activities including, for example, ADCC, CDC, and/orcomplement fixation. This protein may include a binding domainpolypeptide that is a single chain Fv. Additionally, this protein mayinclude a binding domain polypeptide that is a single chain Fv whereinthe heavy chain variable region of the single chain Fv has an amino aciddeletion or substitution at one or more of amino acid positions 9, 10,11, 12, 108, 110, and 112. The protein may also include a binding domainpolypeptide that is a single chain Fv wherein the light chain variableregion of the single chain Fv has an amino acid deletion or substitutionat one or more of amino acid positions 12, 80, 81, 83, 105, 106, and107.

In another aspect, the invention includes a non-naturally occurringV_(H) protein, or a desired portion thereof, that comprises, consistsessentially of, or consists of, alone or in combination with any othermolecule or construct, a V_(H) region or portion thereof that has anamino acid deletion or substitution at one or more of amino acidpositions 9, 10, 11, 12, 108, 110, and 112 of said V_(H) region. Aminoacids may be substituted with either naturally occurring ornon-naturally occurring amino acids, or any other desired usefulmolecule.

Also described and claimed are uses of V_(H) proteins, or desiredportions thereof, that comprise, consist essentially of, or consist of,alone or in combination with any other molecule or construct, a V_(H)region or portion thereof that has an amino acid deletion orsubstitution at one or more of amino acid positions 9, 10, 11, 12, 108,110, and 112 of said V_(H) region. Such uses include uses in phagedisplay, yeast display, and ribosome display systems and methods.

In yet another aspect, the invention includes a non-naturally occurringV_(L) protein, or a desired portion thereof, that comprises, consistsessentially of, or consists of, alone or in combination with any othermolecule, a V_(L) region or portion thereof that has an amino aciddeletion or substitution at one or more of amino acid positions 12, 80,81, 83, 105, 106, and 107 of said V_(L) region. Amino acids may besubstituted with either naturally occurring or non-naturally occurringamino acids, or any other desired useful molecule.

Also described and claimed are uses of V_(L) proteins, or desiredportions thereof, that comprises, consists essentially of, or consistsof, alone or in combination with any other molecule, a V_(L) region orportion thereof that has an amino acid deletion or substitution at oneor more of amino acid positions 12, 80, 81, 83, 105, 106, and 107 ofsaid V_(L) region. Such uses include uses in phage display, yeastdisplay, and ribosome display systems and methods.

In yet another aspect, the invention includes a molecule comprising,consisting essentially of, or consisting of, (1) a V_(H) protein, or adesired portion thereof, wherein the V_(H) protein or portion thereofhas an amino acid deletion or substitution at one or more of amino acidpositions 9, 10, 11, 12, 108, 110, and 112, and (2) a non-naturallyoccurring V_(L) protein, or a desired portion thereof, alone or incombination with any other molecule, wherein the V_(L) protein orportion thereof has an amino acid deletion or substitution at one ormore of amino acid positions 12, 80, 81, 83, 105, 106, and 107. Aminoacids may be substituted with either naturally occurring ornon-naturally occurring amino acids, or any other desired usefulmolecule.

Also described and claimed are uses of a molecule comprising, consistingessentially of, or consisting of, (1) a V_(H) protein, or a desiredportion thereof, wherein the V_(H) protein or portion thereof has anamino acid deletion or substitution at one or more of amino acidpositions 9, 10, 11, 12, 108, 110, and 112, and (2) a non-naturallyoccurring V_(L) protein, or a desired portion thereof, alone or incombination with any other molecule, wherein the V_(L) protein orportion thereof has an amino acid deletion or substitution at one ormore of amino acid positions 12, 80, 81, 83, 105, 106, and 107. Suchuses include uses in phage display, yeast display, and ribosome displaysystems and methods.

The invention also includes molecular constructs wherein the bindingdomain is a single chain Fv and the heavy chain variable region of saidsingle chain Fv has an amino acid substitution at amino acid position11. The amino acid substituted for the amino acid at position of 11 ofthe single chain Fv heavy chain variable region may be selected from thegroup consisting of serine, threonine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, and histidine. Theinvention thus includes, for example, a construct wherein the bindingdomain is a single chain Fv and the heavy chain variable region of saidsingle chain Fv has a serine amino acid substitution at amino acidposition 11. Other amino acid position changes, substitutions, anddeletions, are noted herein.

The invention also includes, for example, a construct wherein thebinding domain is a single chain Fv and the amino acid at position 10and/or 11 of the heavy chain variable region of said single chain Fv hasbeen deleted.

In another aspect, the invention includes constructs wherein the bindingregion binds to a tumor or tumor-associated antigen. The binding regionof a construct of the invention may bind, for example, to a cancer cellantigen. Cancer cell antigens to which constructs of the invention bindinclude cancer cell surface antigens and intracellular cancer cellantigens.

In yet another aspect, the invention includes a construct wherein thebinding region binds to an antigen on an immune effector cell.

In another aspect, the invention includes a construct wherein thebinding region binds to a B cell antigen including, for example, a Bcell antigen selected from the group consisting of CD19, CD20, CD22,CD37, CD40, CD80, and CD86. Constructs of the invention that bind tosuch B cell antigens include, for example, binding regions comprising ansingle chain Fv. Examples of such single chain Fv binding regionsinclude molecules comprising or consisting essentially of single chainFvs selected from the group consisting of HD37 single chain Fv, 2H7single chain Fv, G28-1 single chain Fv, and 4.4.220 single chain Fv.Other examples include a binding region comprising, consistingessentially of, or consisting of, an extracellular domain of CTLA-4.

In another aspect, the invention includes a construct wherein thebinding region binds to a B cell differentiation antigen. B celldifferentiation antigens include, for example, CD19, CD20, CD21, CD22,CD23, CD37, CD40, CD45RO, CD80, CD86, and HLA class II.

In another aspect, the invention includes a construct wherein thebinding region binds to a target selected from the group consisting ofCD2, CD3, CD4, CD5, CD6, CD8, CD10, CD11b, CD14, CD19, CD20, CD21, CD22,CD23, CD24, CD25, CD28, CD30, CD37, CD40, CD43, CD50 (ICAM3), CD54(ICAM1), CD56, CD69, CD80, CD86, CD134 (OX40), CD137 (41BB), CD152(CTLA-4), CD153 (CD30 ligand), CD154 (CD40 ligand), ICOS, L6, B7-H1, andHLA class II.

The invention also includes protein constructs having a binding region,a tail region, and a connecting region, wherein the protein construct iscapable of existing in solution as a monomer or in substantiallymonomeric form.

The invention also includes protein constructs having a binding region,a tail region, and a connecting region, wherein the protein construct iscapable of forming a complex comprising two or more of said proteinconstructs including, for example, wherein said complex is a dimer.

In another aspect, constructs of the invention are capable ofparticipating in or inducing or eliciting or helping to induce orelicit, directly or indirectly, at least one immunological activityselected from the group consisting of antibody dependent cell-mediatedcytotoxicity, complement-dependent cytotoxicity (or complement-mediatedlysis), complement fixation, induction of apoptosis, induction of one ormore biologically active signals, induction of one or more immuneeffector cells, activation of cellular differentiation, cellularactivation, release of one or more biologically active molecules, andneutralization of an infectious agent or toxin.

In another aspect, binding constructs of the invention are capable ofinduction of biologically active signals by activation or inhibition ofone or more molecules selected from the group consisting of proteinkinases, protein phosphatases, G-proteins, cyclic nucleotides or othersecond messengers, ion channels, and secretory pathway components. Suchbiologically active molecules are, for example, proteases. Otherbiologically active molecules are, for example, cytokines, including byway of example monokines, lymphokines, chemokines, growth factors,colony stimulating factors, interferons, and interleukins.

In another aspect, constructs of the invention are capable of induction,or participation in the induction, of one or more immune effector cellsselected from the group consisting of NK cells, monocytes, macrophages,B cells, T cells, mast cells, neutrophils, eosinophils, and basophils.

In another aspect, constructs of the invention are capable of induction,or participation in the induction, of one or more immune effector cellsthat results in antibody dependent cell-mediated cytotoxicity or therelease of one or more biologically active molecules.

In another aspect, constructs of the invention are capable ofparticipating in and/or initiating apopotosis within target cells, forexample, by activating one or more signalling mechanisms or molecules.

In another aspect, constructs of the invention are capable of induction,or participation in the induction, of cellular activation, wherein saidactivation leads to changes in cellular transcriptional activity. In oneembodiment, cellular transcriptional activity is increased. In anotherembodiment, cellular transcriptional activity is decreased.

In another aspect, constructs of the invention having tail regionscomprising, consisting essentially of, or consisting of, constantregions from IgA or IgE molecules, are capable of induction, orparticipation in the induction, of degranulation of neutrophils and/ormast cells.

In another aspect, constructs of the invention are capable of promotion,or participation in the promotion, of neutralization of an infectiousagent, wherein said infectious agent is, for example, a bacterium, avirus, a parasite, or a fungus.

In another aspect, constructs of the invention are capable of promoting,or participating in the promotion of, neutralization of a toxin, whereinsaid toxin is selected from the group consisting of endotoxins andexotoxins. Such toxins include, for example, exotoxins selected from thegroup consisting of anthrax toxin, cholera toxin, diphtheria toxin,pertussis toxin, E. coli heat-labile toxin LT, E. coli heat stable toxinST, shiga toxin Pseudomonas Exotoxin A, botulinum toxin, tetanus toxin,Bordetella pertussis AC toxin, and Bacillus anthracis EF toxin. Othertoxins include, for example, saxitoxins, tetrodotoxin, mushroom toxins(amatoxins, gyromitrin, orellanine, etc.), aflatoxins, pyrrolizidinealkaloids, phytohemagglutinins, and grayanotoxins.

In another aspect, constructs of the invention are capable of binding toan intracellular target to, for example, effect (or participate ineffecting) a cellular function. Such constructs include, for example,constructs that include a tail region comprising, consisting essentiallyof, or consisting of, a native or engineered IgA CH2 domain region and anative or engineered IgA CH3 domain region, said tail region beingcapapble of binding J chain. Such a tail region is found, for example,in the 2H7 scFv IgAH WlgACH2 WCH3+JChain construct. Thus, the inventionincludes constructs having, for example, an “Anti-Intracellular Target”binding domain (for example, and “Anti-Intracellular Target” scFv), aconnecting region, and a native or engineered IgA constant regioncapable of binding J chain (for example, WlgACH2 WCH3).

In still another aspect, constructs of the invention include a moleculewherein an N-terminally immunoglobulin heavy chain constant regionpolypeptide comprises an IgG CH2 constant region polypeptide attached toan immunoglobulin heavy chain IgG CH3 constant region polypeptide.

In yet another aspect, the invention includes a method of reducing atarget cell population in a subject comprising administering to saidsubject a therapeutically effective amount of a protein molecule that isless than about 120 kK, or less than about 150 kD, as measured, forexample, by HPLC and non-reducing gels and consists essentially of (a) afirst protein or peptide molecule that is capable of binding to cellswithin said target cell population, and (b) a second protein or peptidemolecule that is capable of (i) binding to an Fc receptor and/or (ii)inducing target cell apoptosis, and/or (iii) fixing complement, whereinsaid first protein or peptide molecule is directly connected to saidsecond protein or peptide molecule, or, optionally, said first proteinor peptide molecule and said second protein or peptide molecule arelinked by a third protein or peptide molecule, wherein said proteinmolecule is not an antibody, a member of the TNF family or the TNFreceptor family, and is not conjugated with a bacterial toxin, acytotoxic drug, or a radioisotope.

In another aspect, the invention also includes single chain proteinscomprising, consisting essentially of, or consisting of, (i) a firstpolypeptide having a binding domain polypeptide capable of binding to atarget molecule; (ii) a second polypeptide comprising a connectingregion attached to the C-terminus of said first polypeptide; and (iii) athird polypeptide comprising an N-terminally truncated immunoglobulinheavy chain constant region polypeptide attached to the C-terminus ofsaid second polypeptide, wherein said single-chain protein is capable ofat least one immunological activity, and provided that (a) when theconnecting region polypeptide comprises an IgG hinge region polypeptidehaving no cysteine residues, the binding domain polypeptide target isnot CD20 or L6, or (b) when the connecting region polypeptide comprisesan IgG hinge region polypeptide having no cysteine residues, the singlechain protein is not a 1F5 scFv capable of binding to CD20. Theinvention also includes single chain proteins comprising, consistingessentially of, or consisting of, (i) a first polypeptide having abinding domain polypeptide capable of binding to a target molecule; (ii)a second polypeptide comprising a connecting region attached to theC-terminus of said first polypeptide; and (iii) a third polypeptidecomprising an N-terminally truncated immunoglobulin heavy chain constantregion polypeptide attached to the C-terminus of said secondpolypeptide, wherein said single-chain protein is capable of binding toa target molecule on or in a target cell and decreasing the number oftarget cells in vivo and/or depleting a population of target cells invivo. The invention also includes single chain proteins comprising,consisting essentially of, or consisting of, (i) a first polypeptidehaving a binding domain polypeptide capable of binding to a targetmolecule; (ii) a second polypeptide comprising a connecting regionattached to the C-terminus of said first polypeptide; and (iii) a thirdpolypeptide comprising an N-terminally truncated immunoglobulin heavychain constant region polypeptide attached to the C-terminus of saidsecond polypeptide, wherein said single-chain protein is capable ofinducing antibody-dependent cell-mediated cytotoxicity and complementfixation. The invention also includes single chain proteins comprising,consisting essentially of, or consisting of, (i) a first polypeptidehaving a binding domain polypeptide capable of binding to a targetmolecule; (ii) a second polypeptide comprising a connecting regionattached to the C-terminus of said first polypeptide; and (iii) a thirdpolypeptide comprising an N-terminally truncated immunoglobulin heavychain constant region polypeptide attached to the C-terminus of saidsecond polypeptide, wherein said single-chain protein is capable of (1)inducing antibody-dependent cell-mediated cytotoxicity and complementfixation, and (2) binding to a target molecule on or in a target celland decreasing the number of target cells in vivo and/or depleting apopulation of target cells in vivo. The invention also includes singlechain proteins comprising, consisting essentially of, or consisting of,(i) a first polypeptide having a binding domain polypeptide capable ofbinding to a target molecule; (ii) a second polypeptide comprising aconnecting region attached to the C-terminus of said first polypeptide;and (iii) a third polypeptide comprising an N-terminally truncatedimmunoglobulin heavy chain constant region polypeptide attached to theC-terminus of said second polypeptide, wherein when said connectingregion comprises an IgG hinge region polypeptide having at least first,second, and third cysteine residues, said first cysteine beingN-terminal to said second cysteine and said second cysteine beingN-terminal to said third cysteine, one or both of said second and thirdcysteine residues is substituted or deleted, and wherein saidsingle-chain protein is capable of at least one immunological activity.The invention also includes single chain proteins comprising, consistingessentially of, or consisting of, (i) a first polypeptide having abinding domain polypeptide capable of binding to a target molecule; (ii)a second polypeptide comprising a connecting region attached to theC-terminus of said first polypeptide; and (iii) a third polypeptidecomprising an N-terminally truncated immunoglobulin heavy chain constantregion polypeptide attached to the C-terminus of said secondpolypeptide, wherein when said connecting region comprises an IgG hingeregion polypeptide having at least first, second, and third cysteineresidues, said first cysteine being N-terminal to said second cysteineand said second cysteine being N-terminal to said third cysteine, one orboth of said second and third cysteine residues is substituted ordeleted, and wherein said single-chain protein is capable of inducing atleast one immunological activity selected from (a) antibody-dependentcell-mediated cytotoxicity and (b) complement fixation. The inventionalso includes single chain proteins comprising, consisting essentiallyof, or consisting of, (i) a first polypeptide having a binding domainpolypeptide capable of binding to a target molecule; (ii) a secondpolypeptide comprising a connecting region attached to the C-terminus ofsaid first polypeptide; and (iii) a third polypeptide comprising anN-terminally truncated immunoglobulin heavy chain constant regionpolypeptide attached to the C-terminus of said second polypeptide,wherein when said connecting region comprises an IgG hinge regionpolypeptide having at least first, second, and third cysteine residues,said first cysteine being N-terminal to said second cysteine and saidsecond cysteine being N-terminal to said third cysteine, one or both ofsaid second and third cysteine residues is substituted or deleted, andwherein said single-chain protein is capable of antibody-dependentcell-mediated cytotoxicity and complement fixation. The invention alsoincludes single chain proteins comprising, consisting essentially of, orconsisting of, (i) a first polypeptide having a binding domainpolypeptide capable of binding to a target molecule; (ii) a secondpolypeptide comprising a connecting region attached to the C-terminus ofsaid first polypeptide; and (iii) a third polypeptide comprising anN-terminally truncated immunoglobulin heavy chain constant regionpolypeptide attached to the C-terminus of said second polypeptide,wherein when said connecting region comprises an IgG hinge regionpolypeptide having at least first, second, and third cysteine residues,said first cysteine being N-terminal to said second cysteine and saidsecond cysteine being N-terminal to said third cysteine, one or both ofsaid second and third cysteine residues is substituted or deleted, andwherein said single-chain protein is capable of binding to a targetmolecule on or in a target cell and decreasing the number of targetcells in vivo and/or depleting a population of target cells in vivo. Theinvention also includes single chain proteins comprising, consistingessentially of, or consisting of, (i) a first polypeptide having abinding domain polypeptide capable of binding to a target molecule; (ii)a second polypeptide comprising a connecting region attached to theC-terminus of said first polypeptide; and (iii) a third polypeptidecomprising an N-terminally truncated immunoglobulin heavy chain constantregion polypeptide attached to the C-terminus of said secondpolypeptide, wherein when said connecting region comprises an IgG hingeregion polypeptide having at least first, second, and third cysteineresidues, said first cysteine being N-terminal to said second cysteineand said second cysteine being N-terminal to said third cysteine, one orboth of said second and third cysteine residues is substituted ordeleted, and wherein said single-chain protein is capable of inducing atleast one immunological activity selected from antibody-dependentcell-mediated cytotoxicity and complement fixation, and wherein saidsingle-chain protein is capable of binding to a target molecule on or ina target cell and decreasing the number of target cells in vivo and/ordepleting a population of target cells in vivo. The invention alsoincludes single chain proteins comprising, consisting essentially of, orconsisting of, (i) a first polypeptide having a binding domainpolypeptide capable of binding to a target molecule; (ii) a secondpolypeptide comprising a connecting region attached to the C-terminus ofsaid first polypeptide; and (iii) a third polypeptide comprising anN-terminally truncated immunoglobulin heavy chain constant regionpolypeptide attached to the C-terminus of said second polypeptide,wherein when said connecting region comprises an IgG hinge regionpolypeptide having at least first, second, and third cysteine residues,said first cysteine being N-terminal to said second cysteine and saidsecond cysteine being N-terminal to said third cysteine, one or both ofsaid second and third cysteine residues is substituted or deleted, andwherein said single-chain protein is capable of inducingantibody-dependent cell-mediated cytotoxicity and complement fixation,and wherein said single-chain protein is capable of binding to a targetmolecule on or in a target cell and decreasing the number of targetcells in vivo and/or depleting a population of target cells in vivo. Theinvention also includes single chain proteins comprising, consistingessentially of, or consisting of, (i) a first polypeptide having abinding domain polypeptide capable of binding to a target molecule, saidbinding domain polypeptide comprising a heavy chain variable regionwherein leucine at position 11 in the first framework region of theheavy chain variable region is deleted or substituted with another aminoacid; (ii) a second polypeptide comprising a connecting region attachedto the C-terminus of said first polypeptide; and (iii) a thirdpolypeptide comprising an N-terminally truncated immunoglobulin heavychain constant region polypeptide attached to the C-terminus of saidsecond polypeptide, wherein said single-chain protein is capable of atleast one immunological activity, and provided that when the bindingdomain polypeptide is capable of binding to CD20 said connecting regioncomprises three cysteine residues wherein one or two of said threecysteine residues is substituted or replaced with another amino acid.The invention also includes single chain proteins comprising, consistingessentially of, or consisting of, (i) a first polypeptide having abinding domain polypeptide capable of binding to a target molecule, saidbinding domain polypeptide comprising a heavy chain variable regionwherein leucine at position 11 in the first framework region of theheavy chain variable region is deleted or substituted with another aminoacid, (ii) a second polypeptide comprising a connecting region attachedto the C-terminus of said first polypeptide; and (iii) a thirdpolypeptide comprising an N-terminally truncated immunoglobulin heavychain constant region polypeptide attached to the C-terminus of saidsecond polypeptide, wherein said single-chain protein is capable ofinducing at least one immunological activity, provided that when bindingdomain polypeptide is a 2H7 scFv capable of binding to CD20 and saidconnecting region comprises an IgG hinge region polypeptide having atleast first, second, and third cysteine residues, said first cysteinebeing N-terminal to said second cysteine and said second cysteine beingN-terminal to said third cysteine, one or two of said cysteine residuesis substituted or deleted. The invention also includes single chainproteins comprising, consisting essentially of, or consisting of, (i) afirst polypeptide having a binding domain polypeptide capable of bindingto a target molecule on or in a target cell, said binding domainpolypeptide comprising a heavy chain variable region wherein leucine atposition 11 in the first framework region of the heavy chain variableregion is deleted or substituted with another amino acid; (ii) a secondpolypeptide comprising a connecting region attached to said firstpolypeptide; and (iii) a third polypeptide comprising an N-terminallytruncated immunoglobulin heavy chain constant region polypeptideattached to the second polypeptide, wherein said single-chain protein iscapable of at least one immunological activity, provided that saidsingle chain protein does not comprise, or consist essentially of, abinding domain polypeptide capable of binding to CD20 and a connectingregion that comprises (a) an IgG hinge having three cysteine residues or(b) an IgG hinge comprising three serine (or like amino acid) residuesthat have been substituted for cysteine residues. There are manypossible variations and variants of these single chain proteins. Forexample, the binding domain polypeptide may be a single chain antibodyor scFv including naturally occurring and/or non-naturally occurringV_(H) and V_(L) polypeptides, and the binding domain polypeptide maybind any of a number of targets. Non-naturally occurring V_(H)polypeptides include, by way of example and not limitation, human heavychain variable region polypeptide comprising a mutation, substitution,or deletion of an amino acid(s) at a location corresponding to any oneor more of amino acid positions 9, 10, 11, 12, 108, 110, and/or 112.Non-naturally occurring V_(L) polypeptides include, by way of exampleand not limitation, human light chain variable region polypeptidescomprising a mutation, substitution, or deletion of an amino acid(s) ata location corresponding to any one or more of amino acid positions 12,80, 81, 83, 105, 106, and 107. Targets include, by way of example andnot limitation, CD19, CD20, CD28, CD30, CD37, CD40, L6, HER2, epidermalgrowth factor receptors (EGFRs), vascular endothelial cell growthfactors (VEGFs), tumor necrosis factors (e.g., TNF-alpha), as well asother targets described or referred to herein or otherwise useful,whether now known or later discovered. Additionally, the connectingregion polypeptide may be any of a number of molecules, both naturallyoccurring and non-naturally occurring. Connecting region polypeptidesinclude, by way of example and not limitation, naturally occurring andnon-naturally occurring immunoglobulin hinge region polypeptides.Naturally occurring immunoglobulin hinge region polypeptides includewild-type immunoglobulin hinge region polypeptides such as, by way ofexample and not limitation, human IgG1 hinge region polypeptides, humanIgA hinge region polypeptides, human IgD hinge region polypeptides,human IgE hinge-acting regions (e.g., IgE CH2), camelid immunoglobulinhinge regions, or any other naturally occurring hinge region peptidesdescribed or referred to herein or otherwise useful useful, whether nowknown or later discovered. Non-naturally occurring immunoglobulin hingeregion polypeptides include, by way of example and not limitation,mutated naturally occurring immunoglobulin hinges, includingimmunoglobulin hinge region polypeptides that contain less than thewild-type number of cysteines, for example, mutated naturally occurringimmunoglobulin hinge region polypeptides that contain zero, one, or twocysteines, and any other connecting region molecule described orreferenced herein or otherwise useful or now known or later discoveredas useful for connecting joining, for example, immunoglobulin domainssuch as a CH1 domain and a CH2 domain. N-terminally truncatedimmunoglobulin heavy chain constant region polypeptides includenaturally occurring and non-naturally occurring N-terminally truncatedimmunoglobulin heavy chain constant region polypeptides that, with orwithout other portions of the single chain protein, provide one or moreeffector functions such as those described herein. Naturally occurringN-terminally truncated immunoglobulin heavy chain constant regionpolypeptides include, by way of example and not limitation, CH2CH3constant region polypeptides, including CH2CH3 constant regionpolypeptides taken, separately or together, from human IgGs, human IgAs,and human IgE, and any other immunoglobulin heavy chain constant regionpolypeptide described or referenced herein or otherwise now known orlater discovered to be useful. Non-naturally occurring N-terminallytruncated immunoglobulin heavy chain constant region polypeptidesinclude, by way of example and not limitation, any mutated naturallyoccurring heavy chain constant region polypeptide described or referredto herein or otherwise now known or later discovered to be useful.

Various specific constructs of the invention include, by way of exampleonly, the following:

-   -   1. 2H7 scFv VH L11S (CSC-S) H WCH2 WCH3    -   2. 2H7 scFv VH L11S IgE CH2 CH3 CH4    -   3. 2H7 scFv VH L11S mIgE CH2 CH3 CH4    -   4. 2H7 scFv VH L11S mIgAH WIgACH2 T4-CH3    -   5. 2H7 scFv VH L11S (SSS-S) H K322S CH2 WCH3    -   6. 2H7 scFv VH L11S (CSS-S) H K322S CH2 WCH3    -   7. 2H7 scFv VH L11S (SSS-S) H P331S CH2 WCH3    -   8. 2HU scFv VH L11S (CSS-S) H P331S CH2 WCH3    -   9. 2H7 scFv VH L11S (SSS-S) H T256N CH2 WCH3    -   10. 2H7 scFv VH L11S (SSS-S) H RTPE/QNAK (255-258) CH2 WCH3    -   11. 2H7 scFv VH L11S (SSS-S) H K290Q CH2 WCH3    -   12. 2H7 scFv VH L11S (SSS-S) H A339P CH2 WCH3    -   13. G28-1 scFv (SSS-S) H WCH2 WCH3    -   14. G28-1 scFv IgAH WCH2 WCH3    -   15. G28-1 scFv VH L11S (SSS-S) H WCH2 WCH3    -   16. G28-1 scFv VH L11S (CSS-S) H WCH2 WCH3    -   17. G28-1 scFv VH L11S(CSC-S) H WCH2 WCH3    -   18. G28-1 scFv VH L11S (SSC-P) H WCH2 WCH3    -   19. CTLA4 (SSS-S) H P238SCH2 WCH32    -   20. CTLA4 (CCC-P) WH WCH2 WCH3    -   21. FC2-2 scFv (SSS-S) H WCH2 WCH3    -   22. FC2-2 scFv VHL11S (SSS-S) H WCH2 WCH3    -   23. UCHL-1 scFv (SSS-S) H WCH2 WCH3    -   24. UCHL-1 scFv VHL11S (SSS-S) H WCH2 WCH3    -   25. 5B9 scFv (SSS-S) H WCH2 WCH3    -   26. 5B9 scFv VHL11S (SSS-S) H WCH2 WCH3    -   27. 2H7 scFv (SSS-S) H WCH2 WCH3    -   28. 2H7 scFv (SSS-S) H P238SCH2 WCH3    -   29. 2H7 scFv IgAH WCH2 WCH3    -   30. 2H7 scFv IgAH WIgACH2 T4-CH3    -   31. 2H7 scFv IgAH WlgACH2 WCH3+JChain    -   32. 2H7 scFv (CCC-P) WH WCH2 WCH3    -   33. 2H7 scFv (SSS-S) H WCH2 F405YCH3    -   34. 2H7 scFv (SSS-S) H WCH2 F405ACH3    -   35. 2H7 scFv (SSS-S) H WCH2 Y407ACH3    -   36. 2H4 scFv (SSS-S) HWCH2 F405A, Y407ACH3    -   37. 2H7 scFv (CSS-S) H WCH2 WCH3    -   38. 2H7 scFv (SCS-S) H WCH2 WCH3    -   39. 2H7 scFv (SSC-P) H WCH2 WCH3    -   40. 2H7 scFv (CSC-S) H WCH2 WCH3    -   41. 2H7 scFv (CCS-P) H WCH2 WCH3    -   42. 2H7 scFv (SCC-P) H WCH2 WCH3    -   43. 2H7 scFv VH L11S (SSS-S) H WCH2 WCH3    -   44. 2H7 scFv VH L11S (CSS-S) H WCH2 WCH3    -   45. G28-1 scFv VH L11S (SCS-S) H WCH2 WCH3    -   46. G28-1 scFv VH L11S(CCS-P) H WCH2 WCH3    -   47. G28-1 scFv VH L11S (SCC-P) H WCH2 WCH3    -   48. G28-1 scFv VH L11S mlgE CH2 CH3 CH4    -   49. G28-1 scFv VH L11S mIgAH WIgACH2 T4CH3    -   50. G28-1 scFv VH L11S hIgE CH2 CH3 CH4    -   51. G28-1 scFv VH L11S hIgAH WIgACH2 T4CH3    -   52. HD37 scFv IgAH WCH2 WCH3    -   53. HD37 scFv (SSS-S) H WCH2 WCH3    -   54. HD37 scFv VH L11S (SSS-S) H WCH2 WCH3    -   55. L6 scFv lgAH WCH2 WCH3    -   56. L6 scFv VHL11S (SSS-S) H WCH2 WCH3    -   57. 2H7 scFv-llama IgG1    -   58. 2H7 scFv-llama IgG2    -   59. 2H7 scFv-llama IgG3    -   60. CD16-6 low (ED)(SSS-S) H P238SCH2 WCH3    -   61. CD16-9 high (ED)(SSS-S) H P238SCH2 WCH3    -   62. 2e12 scFv (SSS-s)H P238SCH2 WCH3-hCD80TM/CT    -   63. 10A8 scFv (SSS-s)H P238SCH2 WCH3-hCD80TM/CT    -   64. 40.2.36 scFv (SSS-s)H P238SCH2 WCH3-hCD80TM/CT    -   65. 2H7 scFv (SSS-s)H P238SCH2 WCH3-hCD80TM/CT    -   66. G19-4 scFv (SSS-s)H P238SCH2 WCH3-hCD80TM/CT    -   67. 2e12 scFv (SSS-s)H WCH2 WCH3-hCD80TM/CT    -   68. 2e12 scFv IgAH IgACH2 T4CH3-hCD80TM/CT    -   69. 2e12 scFv IgE CH2CH3CH4-hCD80TM/CT    -   70. 2e12 scFv (SSS-s)H P238SCH2 WCH3-mFADD-TM/CT    -   71. 2e12 scFv (SSS-s)H WCH2 WCH3-mFADD-TM/CT    -   72. 2e12 scFv (SSS-s)H WCH2 WCH3-mcasp3-TM/CT    -   73. 2e12 scFv (SSS-s)H P238SCH2 WCH3-mcasp3-TM/CT    -   74. 2e12 scFv (SSS-s)H WCH2 WCH3-mcasp8-TM/CT    -   75. 2e12 scFv (SSS-s)H P238SCH2 WCH3-mcasp-8-TM/CT    -   76. 2e12 scFv (SSS-s)H WCH2 WCH3-hcasp3-TM/CT    -   77. 2e12 scFv (SSS-s)H P238SCH2 WCH3-hcasp3-TM/CT    -   78. 2e12 scFv (SSS-s)H WCH2 WCH3-hcasp8-TM/CT    -   79. 2e12 scFv (SSS-s)H P238SCH2 WCH3-hcasp8-TM/CT    -   80. 1D8 scFv-hIgG1 (SSS-s)H P238SCH2 WCH3-hCD80TM/CT    -   81. 1D8 scFv-hIgG1 (SSS-s)H WCH2 WCH3-hCD80TM/CT    -   82. 1D8 scFv-mIgAT4-hCD80TM/CT    -   83. 1D8 scFv-hIgE-hCD80TM/CT    -   84. 1D8 scFv-hIgG1 (SSS-s)H P238SCH2 WCH3-mFADD-TM/CT    -   85. 1D8 scFv-hIgG1 (SSS-s)H WCH2 WCH3-mFADD-TM/CT    -   86. 1D8 scFv-hIgG1 (SSS-s)H WCH2 WCH3-mcasp3-TM/CT    -   87. 1D8 scFv-hIgG1 (SSS-s)H P238SCH2 WCH3-mcasp3-TM/CT    -   88. 1D8 scFv-hIgG1 (SSS-s)H WCH2 WCH3-mcasp8-TM/CT    -   89. 1D8 scFv-hIgG1 (SSS-s)H P238SCH2 WCH3-mcasp8-TM/CT    -   90. 1D8 scFv-hIgG1 (SSS-s)H WCH2 WCH3-hcasp3-TM/CT    -   91. 1D8 scFv-hIgG1 (SSS-s)H P238SCH2 WCH3-hcasp3-TM/CT    -   92. 1D8 scFv-hIgG1 (SSS-s)H WCH2 WCH3-hcasp8-TM/CT    -   93. 1D8 scFv-hIgG1 (SSS-s)H P238SCH2 WCH3-hcasp8-TM/CT L6 scFv        (SSS-S) H WCH2 WCH3    -   94. 2H7 scFv CD154 (L2)    -   95. 2H7 scFv CD154 (S4)    -   96. CTLA4 IgAH IGACH2CH3    -   97. CTLA4 IgAH IgACH2 T4CH3    -   98. 2H7 scFv IgAH IgACH2CH3    -   99. 2H7 scFv IgAH IgAHCH2 T18CH3    -   100. 2H&-40.2.220 scFv (SSS-S) H WCH2 WCH3 (bispecific        anti-ccd20-anti-cd40)    -   101. 2H7 scFv IgAH IgACH2 T4CH3-hCD89 TM/CT    -   102. G19-4 scFv (CCC-P) WH WCH2 WCH3-hCD89 TM/CT    -   103. 2e12 scFv (CCC-P) WH WCH2 WCH3-hCD89 TM/CT

These and other aspects of the present invention will become furtherapparent upon reference to the following detailed description andattached drawings. As noted herein, all referenced patents, articles,documents, and other materials disclosed or identified herein are herebyincorporated by reference in their entireties as if each wasincorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows DNA and deduced amino acid sequences [SEQ ID NOS:688 and689] of 2H7 scFv-Ig, a binding domain-immunoglobulin fusion proteincapable of specifically binding CD20.

FIG. 2 shows production levels of 2H7 scFv-Ig by transfected, stable CHOlines and generation of a standard curve by binding of purified 2H7scFv-Ig to CHO cells expressing CD20.

FIG. 3 shows SDS-PAGE analysis of multiple preparations of isolated2H7scFv-Ig protein.

FIG. 4 shows complement fixation (FIG. 4A) and mediation ofantibody-dependent cellular cytotoxicity (FIG. 4B) by 2H7scFv-Ig.

FIG. 5 shows the effect of simultaneous ligation of CD20 and CD40 ongrowth of normal B cells.

FIG. 6 shows the effect of simultaneous ligation of CD20 and CD40 onCD95 expression and induction of apoptosis in a B lymphoblastoid cellline.

FIG. 7 shows DNA and deduced amino acid sequences of 2H7scFv-CD154 L2(FIG. 7A, SEQ ID NOS:690 and 691) and 2H7scFv-CD154 S4 (FIG. 7B, SEQ IDNOS:692 and 693) binding domain-immunoglobulin fusion proteins capableof specifically binding CD20 and CD40.

FIG. 8 shows binding of 2H7scFv-CD154 binding domain-immunoglobulinfusion proteins to CD20+CHO cells by flow immunocytofluorimetry.

FIG. 9 shows binding of Annexin V to B cell lines Ramos, BJAB, and T51after binding of 2H7scFv-CD154 binding domain-immunoglobulin fusionprotein to cells.

FIG. 10 shows effects on proliferation of B cell line T51 followingbinding of 2H7scFv-CD154 binding domain-immunoglobulin fusion protein.

FIG. 11 depicts schematic representations of the structures of2H7ScFv-Ig fusion proteins referred to as CytoxB or CytoxB derivatives:CytoxB-MHWTG1C (2H7 ScFv, mutant hinge, wild-type human IgG1 Fc domain),CytoxB-MHMG1C (2H7 ScFv, mutant hinge, mutated human IgG1 Fc domain) andCytoxB-IgAHWTHG1C (2H7 ScFv, human IgA-derived hinge [SEQ ID NO:41],wild-type human IgG1 Fc domain). Arrows indicate position numbers ofamino acid residues believed to contribute to FcR binding and ADCCactivity (heavy arrows), and to complement fixation (light arrows). Noteabsence of interchain disulfide bonds. Also shown are a peptide linker(SEQ ID NO:682) and a portion of the human IgA hinge (SEQ ID NO:683).

FIG. 12 shows SDS-PAGE analysis of isolated CytoxB and 2H7scFv-CD154binding domain-immunoglobulin fusion proteins.

FIG. 13 shows antibody dependent cell-mediated cytotoxicity activity ofCytoxB derivatives.

FIG. 14 shows complement dependent cytotoxicity of CytoxB derivatives.

FIG. 15 shows serum half-life determinations of CytoxB-MHWTG1C inmacaque blood samples.

FIG. 16 shows effects of CytoxB-MHWTG1C on levels of circulating CD40+Bcells in macaque blood samples.

FIG. 17 shows production levels of HD37 (CD19-specific) ScFv-Ig bytransfected mammalian cell lines and generation of a standard curve bybinding of purified HD37 ScFv-Ig to cells expressing CD19.

FIG. 18 shows production levels of L6 (carcinoma antigen) ScFv-Ig bytransfected, stable CHO lines and generation of a standard curve bybinding of purified L6 ScFv-Ig to cells expressing L6 antigen.

FIG. 19 shows antibody dependent cell-mediated cytotoxicity activity ofbinding domain-immunoglobulin fusion proteins 2H7 ScFv-Ig, HD37 ScFv-Igand G28-1 (CD37-specific) ScFv-Ig.

FIG. 20 shows antibody dependent cell-mediated cytotoxicity activity ofL6 ScFv-Ig fusion proteins.

FIG. 21 shows SDS-PAGE analysis of L6 ScFv-Ig and 2H7 ScFv-Ig fusionproteins.

FIG. 22 shows SDS-PAGE analysis of G28-1 ScFv-Ig and HD37 ScFv-Ig fusionproteins.

FIG. 23 presents a sequence alignment of immunoglobulin hinge and CH2domains of human IgG1 (SEQ ID NO:684) with the hinge and CH2 domains ofllama IgG1 (SEQ ID NO:685), IgG2 (SEQ ID NO:686), and IgG3 (SEQ IDNO:687).

FIG. 24 illustrates migration of purified 2H7 scFv llama IgG fusionproteins in a 10% SDS polyacrylamide gel. Purified fusion proteins (5 μgper sample) were prepared in non-reducing sample buffer (lanes 2-5) andin reducing sample buffer (lanes 6-9). Lane 1: molecular weight markers(non-reduced); lanes 2 and 6: 2H7 scFv-llama IgG1 (SEQ ID NOS:21 and22); Lanes 3 and 7: 2H7 scFv-llama IgG2 (SEQ ID NOS:23 and 24): lanes 4and 8: 2H7 scFv-llama IgG3 (SEQ ID NOS:25 and 26); and Lanes 5 and 9:Rituximab (chimeric anti-CD20 antibody (human IgG1 constant region)).

FIG. 25 shows binding of 2H7 scFv-llama IgG1 (SEQ ID NOS:21 and 22), 2H7scFv-llama IgG2 (SEQ ID NOS:23 and 24), and 2H7 scFv-llama IgG3 (SEQ IDNOS:25 and 26) to CD20+CHO cells detected by flow immunocytofluorimetry.

FIG. 26 depicts CDC activity of 2H7 scFv llama IgG fusion proteins, 2H7scFv-llama IgG1 (SEQ ID NO:22), 2H7 scFv-llama IgG2 (SEQ ID NO:24), and2H7 scFv-llama IgG3 (SEQ ID NO:26), and 2H7 scFv human IgG1 (2H7 scFvIgG WTH WTCH2CH3) (SEQ ID NO:28) against BJAB cells in the presence ofrabbit complement. Rituximab was included as a control.

FIG. 27 shows antibody dependent cell-mediated cytotoxicity activity of2H7 scFv llama IgG fusion proteins, 2H7 scFv-llama IgG1 (SEQ ID NO:22),2H7 scFv-llama IgG2 (SEQ ID NO:24), and 2H7 scFv-llama IgG3 (SEQ IDNO:26). Effector cells (human PBMC) were combined with target cells(BJAB cells) at three different ratios, 1:25, 1:50, and 1:100. Rituximabwas included as a control. Each data point represents three separatemeasurements.

FIG. 28 shows antibody dependent cell-mediated cytotoxicity activity of2H7 scFv llama IgG fusion proteins, 2H7 scFv-llama IgG1 (SEQ ID NO:22),2H7 scFv-llama IgG2 (SEQ ID NO:24), and 2H7 scFv-llama IgG3 (SEQ IDNO:26). Effector cells (llama PBMC) were combined with target cells(BJAB cells) at three different ratios, 1:25, 1:50, and 1:100. Rituximabwas included as a control. Each data point represents three separatemeasurements.

FIG. 29 depicts complement dependent cytotoxicity activity of Reh cells(acute lymphocytic leukemia) expressing scFv-Ig fusion proteins on thecell surface. Reh cells were transfected with constructs encoding scFvantibodies specific for human costimulatory molecules, CD152, CD28,CD40, and CD20, fused to human IgG1 wild-type hinge-CH2-CH3, which wasfused to human CD80 transmembrane and cytoplasmic tail domains.Complement dependent cytotoxicity activity was measured in the presenceand absence of rabbit complement (plus C′ and no C′, respectively). Thedata represent the average of duplicate samples. Reh anti-hCD152 scFvIg:Reh cells transfected with polynucleotide 10A8 scFv IgG MTH (SSS) MTCH2CH3 (SEQ ID NO:53); Reh anti-hCD28scFvIg: 2E12 scFv IgG MTH (SSS) MTCH2CH3 (SEQ ID NO:51); Reh anti-hCD40scFvIg: 4.2.220 scFv IgG MTH (SSS)MT CH2CH3 (SEQ ID NO:49); and Reh anti-hCD20scFvIg: 2H7 scFv IgG MTH(SSS) MT CH2CH3 (SEQ ID NOS:57 and 29).

FIG. 30 presents antibody dependent cell-mediated cytotoxicity activityof Reh cells that were transfected with constructs encoding scFvantibodies specific for human costimulatory molecules, CD152, CD28,CD40, and CD20, as described for FIG. 29, and for murine CD3, fused tohuman mutant IgG1 hinge and mutant CH2 and wild type CH3 (Rehanti-mCD3scFv designating Reh cells transfected with polynucleotide500A2 scFv IgG MTH (SSS) MTCH2WTCH3 SEQ ID NO:55)), which was fused tohuman CD80 transmembrane and cytoplasmic tail domains. The datarepresent the average of quadruplicate samples.

FIG. 31 lists immunoglobulin constant region constructs that were usedin experiments illustrated in subsequent figures.

FIG. 32 depicts complement dependent cytotoxicity activity of CTLA-4 Igfusion proteins, CTLA-4 IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:_) (2 μg/ml)and CTLA-4 IgG MTH MTCH2WTCH3 (SEQ ID NO:_) (2 μg/ml), in the presenceand absence of rabbit complement (plus C′ and no C′, respectively). Thetarget cells were Reh cells and Reh cells transfected with CD80 (RehCD80.10).

FIG. 33 shows antibody dependent cell-mediated cytotoxicity activity ofCTLA-4 Ig fusion proteins, CTLA-4 IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:78)(2 μg/ml) and CTLA-4 IgG MTH MTCH2WTCH3 (SEQ ID NOS:530 and 86) (2μg/ml). Effector cells, human PBMC, were added to target cells, Reh orReh CD80.1, at the ratios indicated. FIG. 33A presents the level ofnatural killing in Reh CD80.1 cells in the absence of any Ig fusionprotein. FIG. 33B presents antibody dependent cell-mediated cytotoxicitymediated by CTLA-4 IgG MTH MTCH2WTCH3, and FIG. 33C presents antibodydependent cell-mediated cytotoxicity mediated by CTLA-4 IgG WTH (CCC)WTCH2CH3. Each data point represents the average percent specifickilling measured in four sample wells.

FIG. 34 illustrates binding of 2H7 (anti-CD20) scFv Ig fusion proteinsto (CD20+) CHO cells by flow immunocytofluorimetry.

FIG. 35 presents an immunoblot of 2H7 scFv IgG and IgA fusion proteins.COS cells were transiently transfected with various 2H7 scFv Ig fusionprotein constructs. The expressed polypeptides were immune precipitatedwith protein A, separated in a non-reducing SDS polyacrylamide gel, andthen transferred to a polyvinyl fluoride membrane. Proteins weredetected using an anti-human IgG (Fc specific) horseradish peroxidaseconjugate. Lane 1: vector only; lane 2: 2H7 scFv IgG WTH (CCC) WTCH2CH3(SEQ ID NO:28); lane 3: 2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID NO:135);lane 4: 2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQ ID NO:137); lane 5: 2H7scFv IgAH IgG WTCH2CH3 (SEQ ID NO:60); and lane 6: 2H7 scFv IgG MTH(SSS) WTCH2CH3 (SEQ ID NO:50).

FIG. 36 illustrates binding of 2H7 scFv IgAH IgACH2CH3 polypeptide (SEQID NO:62) and 2H7 scFv IgAH IgAT4 (SEQ ID NO:71) to (CD20+) CHO cells byflow immunocytofluorimetry. The source of the polypeptides was culturesupernatants from transiently transfected COS cells. COS cellstransfected with a plasmid comprising a sequence encoding 2H7 scFv IgAHIgACH2CH3 were co-transfected with a plasmid containing nucleotidesequence encoding human J chain.

FIG. 37 illustrates antibody dependent cell-mediated cytotoxicityactivity of anti-CD20 (2H7) scFv Ig fusion proteins against BJAB targetcells using whole blood as the source of effector cells. Purified 2H7scFv Ig fusion proteins were titrated and combined with ⁵¹Cr-labeledBJAB cells (5×10⁴) and whole blood (1:4 final dilution). Each data pointrepresents the average percent specific killing measured in four samplewells.

FIG. 38 demonstrates antibody dependent cell-mediated cytotoxicityactivity of 2H7 scFv Ig fusion proteins (5 μg/ml) against ⁵¹Cr-labeledBJAB cells at 0.25, 0.125, and 0.625 dilutions of whole blood. Each datapoint represents the average percent specific killing measured in foursample wells.

FIG. 39 shows a comparison of antibody dependent cell-mediatedcytotoxicity activity of 2H7 scFv IgG MTH (SSS) WTCH2CH3 (5 μg/ml) and2H7 scFv IgAH IgACH2CH3 (5 μg/ml) when human PBMC are the source ofeffector cells (FIG. 39A) and when human whole blood is the source ofeffector cells (FIG. 39B).

FIG. 40 presents an immunoblot of 2H7 scFv IgG fusion proteins. COScells were transiently transfected with various 2H7 scFv Ig fusionprotein constructs. Culture supernatants containing the expressedpolypeptides were separated in a non-reducing SDS polyacrylamide gel,and then were transferred to a polyvinyl fluoride membrane. Proteinswere detected using an anti-human IgG (Fc specific) horseradishperoxidase conjugate. Lanes 1-5: purified 2H7 scFv IgG MTH (SSS)WTCH2CH3 at 40 ng, 20 ng, 10 ng/5 ng, and 2.5 ng per lane, respectively.Culture supernatants were separated in lanes 6-9. Lane 6: 2H7 scFv IgGWTH (CCC) WTCH2CH3; lane 7: 2H7 scFv IgG MTH (CSS) WTCH2CH3; lane 8: 2H7scFv IgG MTH (SCS) WTCH2CH3; and lane 9: 2H7 scFv VHSER11 IgG MTH (SSS)WTCH2CH3. The molecular weight (kDal) of marker proteins is indicated onthe left side of the immunoblot.

FIG. 41 illustrates cell surface expression of 1D8 (anti-murine 4-1BB)scFv IgG WTH WTCH2CH3-CD80 fusion protein on K1735 melanoma cells byflow immunofluorimetry (FIG. 41A). The scFv fusion protein was detectedwith phycoerythrin-conjugated F(ab′)₂ goat anti-human IgG. FIG. 41Bdepicts growth of tumors in naïve C3H mice transplanted by subcutaneousinjection with wild type K1735 melanoma cells (K1735-WT) or with K1735cells transfected with 1D8 scFv IgG WTH WTCH2CH3-CD80 (K1735-1D8). Tumorgrowth was monitored by measuring the size of the tumor. FIG. 41Cdemonstrates the kinetics of tumor growth in naïve C3H mice injectedintraperitoneally with monoclonal antibodies to remove CD8⁺, CD4⁺, orboth CD4⁺ and CD8⁺ T cells prior to transplantation of the animals withK1735-1D8 cells.

FIG. 42 demonstrates therapy of established K1735-WT tumors usingK1735-1D8 as an immunogen. Six days after mice were transplanted withK1735-WT tumors, one group (five animals) was injected subcutaneouslywith K1735-1D8 cells (open circles) or irradiated K1735-WT cells (solidsquares) on the contralateral side. A control group of mice received PBS(open squares). Treatments were repeated on the days indicated by thearrows.

FIG. 43 shows the growth of tumors in animals that were injectedsubcutaneously with 2×10⁶ K1735-WT cells (solid squares) and the growthof tumors in animals that were injected subcutaneously with 2×10⁶K1735-WT cells plus 2×10⁵ K1735-1D8 cells (open triangles).

FIG. 44 presents a flow cytometry analysis of antigen104 murine sarcomatumor cells transfected with 1D8 scFv IgG WTH WTCH2CH3-CD80 isolatedafter repeated rounds of panning against anti-human IgG. Transfectedcells expressing 1D8 scFv IgG WTH WTCH2CH3-CD80 were detected withfluoroisothiocyanate (FITC)-conjugated goat anti-human IgG (depicted inblack). Untransfected cells are shown in gray.

FIG. 45 illustrates migration of various 2H7 scFv Ig fusion proteins ina 10% SDS-PAGE gel. 2H7 was the anti-CD20 scFv and 40.2.220 was theanti-CD40 scFv. Lane 1: Bio-Rad prestained molecular weight standards;lane 2: anti-CD20 scFv IgG MTH (SSS) MTCH2WTCH3; lane 3: anti-CD20 scFvIgG MTH (SSS) WTCH2CH3; lane 4: 2H7 scFv IgAH IgG WTCH2CH3; lane 5:anti-CD20-anti-CD40 scFv IgG MTH (SSS) MTCH2WTCH3; lane 6: Rituximab;lane 7: Novex Multimark® molecular weight standards.

FIG. 46 illustrates effector function as measured in an antibodydependent cell-mediated cytotoxicity assay of 2H7 Ig fusion proteinsthat contain a mutant CH2 domain or wild type CH2 domain. The percentspecific killing of BJAB target cells in the presence of human PBMCeffector cells by 2H7 scFv IgG MTH (SSS) MTCH2WTCH3 (diamonds) wascompared to 2H7 scFv IgG MTH (SSS) WTCH2CH3 (squares) and 2H7 scFv IgAHIgG WTCH2CH3 (triangles) and Rituximab (circles).

FIG. 47 shows cell surface expression of an anti-human CD3 scFv IgG WTHWTCH2CH3-CD80 (SEQ ID NO:413) fusion protein on Reh cells (FIG. 47A) andT51 lymphoblastoid cells (FIG. 47B) by measuring the linear fluorecentequivalent (LFE) using flow immunocytofluorimetry.

FIG. 48 presents the percent specific killing of untransfected Reh andT51 cells and the percent specific killing of Reh cells (Reh anti-hCD3)(FIG. 48A) and T51 cells (T51 anti-hCD3) (FIG. 48B) that weretransfected with a construct encoding scFv antibodies specific for humanCD3, fused to human IgG1 wild-type hinge-CH2-CH3, which was fused tohuman CD80 transmembrane and cytoplasmic tail domains (anti-human CD3scFv IgG WTH WTCH2CH3-CD80 (SEQ ID NO:29). Human PBMC (effector cells)were combined with BJAB target cells at the ratios indicated.

FIG. 49 illustrates binding of 5B9, an anti-murine CD137 (4-1BB)monoclonal antibody, and a 5B9 scFv IgG fusion protein (5B9 scFv IgG MTH(SSS) WTCH2CH3 (SEQ ID NO:133) to stimulated human PBMC. Binding of the5B9 scFv IgG fusion protein was detected by flow immunocytofluorimetryusing FITC conjugated goat anti-human IgG. Binding of the 5B9 monoclonalantibody was detected with FITC conjugated goat anti-mouse IgG.

FIG. 50 illustrates the effect of the LV_(H)11S mutation on theexpression of 2H7 LV_(H)11S scFv WCH2 WCH3 (“CytoxB scFv Ig”; SEQ IDNO:369) in CHO cell lines.

FIG. 51 shows a semi-quantitative SDS-PAGE analysis examining theexpression of 2H7 LV_(H)11S scFv WCH2 WCH3 (SEQ ID NO:369) whentransiently transfected in CHO cells. Lanes 2-5 are various amounts of2H7 LV_(H)11S scFv WCH2 WCH3. Lanes 6-10 are 10 μl samples from fivedifferent clones expressing 2H7 LV_(H)11S scFv WCH2 WCH3.

FIG. 52 shows differences in binding capacity between a G28-1 LV_(H)11SscFv Ig construct (SEQ ID NO:324) and a G28-1 wild type scFv Ig bindingdomain fusion protein construct (SEQ ID NO:320), both obtained fromtransiently transfected COS cells. Binding to Ramos cells was determinedusing flow cytometry. The data illustrates a significant increase inbinding of the V_(H)11S protein to CD37+Ramos cells.

FIG. 53 illustrates increased levels of expression of a G28-1 LV_(H)11SscFv Ig construct (SEQ ID NO:324) compared to a G28-1 wild type scFv Igconstruct in COS. Protein levels were compared using immunoblotanalysis. Both immunoblot gels have quantitated amounts of purified aG28-1 scFv Ig (SSS-S) H WCH2 WCH3 contruct of the invention in lanes1-4. Lanes 5-9 of the first immunoblot represent five different cloneseach transfected with G28-1 scFv (SSS-S) H WCH2 WCH3, while lanes 5-9 ofthe second immunoblot represent five different clones transfected withG28-1 LV_(H)11S scFv (SSS-S) H WCH2 WCH3. The immunoblots illustratethat the LV_(H)11S form causes the G28-1 scFv Ig construct to express atvery high levels.

FIG. 54 illustrates the binding of 2H7 scFv Ig derivatives with alteredhinges) to CHO cells expressing CD20 (CD20+CHO) by flow cytometry, andindicates that these altered connecting region hinge constructs(including (SSS-S), (CSS-S), (SCS-S) and (CSC-S) hinge regions) retainbinding function to CD20.

FIG. 55 shows the ability to mediate antibody dependent cell-mediatedcytotoxicity of various constructs against Bjab targets: (A) 2H7 scFv Igconstructs of the invention that contain connecting regions comprising(CSS-S), (SCS-S), (CSC-S), and (SSS-S) hinges and (B) 2H7 scFvconstructs of the invention with various connecting regions and tailregions. Percent specific killing is compared to total killing inducedby a detergent. The controls are natural killing in target cells witheffectors added and a 2H7 construct with an IgA hinge connecting regionand IgA-derived tail region that does not bind PBMC effectors.

FIG. 56 illustrates the ability of various 2H7 scFv Ig constructs of theinvention that include connecting regions having various hinge regions(e.g., (CSC-S), (SSS-S), (SCS-S), and (CSS-S)) to mediate complementactivity in Ramos cells. Percent specific killing is measured againstthe control of complement only, and 100% killing was determined byexposure of cells to detergent.

FIG. 57 illustrates the shows the binding of 2H7 scFv Ig constructs ofthe invention containing different tail regions to CD20+CHO usingimmunocytofluoroimetry. The different proteins were detected using FITCconjugated to anti-IgG, anti-IgA, and anti-IgE.

FIG. 58A shows the binding of 2H7 V_(H) L11S scFv IgECH2CH3CH4, purifiedusing Hydrophobic charge induction chromatography (HCIC) and eluted atdifferent pHs 4.0 and 3.5, in CD20+CHO cells by flow cytometry,indicating that the proteins bound CD20 whether eluted at pH 4.0 or 3.5.FIG. 58B is a data graph indicating the ability of these 2H7 VH L11SscFv IgE constructs of the invention to mediate, for example, ADCC inBjab target cells.

FIG. 59 shows the binding capacity of G28-1 VH L11S mIgECH2CH3CH4 (A) toBjab and Ramos target cells and (B) to CD20+CHO cells by flow cytometry.

FIG. 60 shows the High Performance Liquid Chromatography (HPLC) profilesof various protein constructs of the invention (A) 2H7 scFv (SSS-S) H(P238S)CH2 WCH3 (B) 2H7 scFv (CSS-S) H WCH2 WCH3, (C) 2H7 scFv (SCS-S) HWCH2 WCH3, and (D) 2H7 scFv (SSS-S) H WCH2 (Y407A)CH3, indicating thatconstruct A has apparent molecular weight forms of 100 kD and 75 kD andthat, by introducing certain changes a predominant 75 kD molecularweight form is obtained, as seen in constructs B, C, and D. See Example40.

FIG. 61 shows the HPLC profiles of various protein constructs of theinvention (A) 2H7 scFv (SSS-S) H WCH2 WCH3, (B) 2H7 scFv (CSC-S) H WCH2WCH3, (C) 2H7 scFv (CCC-P) H WCH2 WCH3, and (D) 2H7 scFv IgAH WCH2 WCH3,indicating that construct A has apparent molecular weight forms of 100kD and 75 kD and that, by introducing certain changes a predominant 75kD molecular weight form is obtained, as seen in constructs B and C,while construct D (which has an IgA tail regaion) has an apparentmolecular weight of 150 kD. See Example 40.

FIG. 62 shows the HPLC profiles of various protein constructs of theinvention (A) 2H7 scFv (SSS-S) H WCH2 WCH3, (B) 2H7 scFv (SCS-S) H WCH2WCH3, (C) 2H7 scFv IgA 3TCH2 WCH3, and (D) 2H7 scFv (SSS-S) H WCH2(F405A Y407A)CH3, indicating that construct A has two forms withapparent molecular weights at 100 kD and 75 kD, construct B has apredominant form with an apparent molecular weight of 75 kD, whileconstruct C with a T4 mutation leads to three forms with apparentmolecular weights near 600 kD and construct D with a double pointmutation in the CH3 region leads to a predominant form having anapparent molecular weight less than 44 kD. A T4 mutation here refers toa truncation of four amino acids from a CH3 region. See Example 40.

FIG. 63 compares the effect on binding CD20+CHO cells by 2H7 V_(H) L11SscFv Ig constructs, with and without F405A and Y407A alterations in theCH3 region, by flow cytometry, indicating a loss of binding capabilitywith this double amino acid change. See Example 41.

FIG. 64 shows the binding capacity of FITC conjugated 2H7 V_(H) L11SscFv Ig derivatives to CH20+CHO cells by flow cytometry, indicating thatthese constructs do not lose binding capacity when conjugated to aflorescent marker. See Example 41.

FIG. 65 shows a nonreducing SDS-PAGE analysis examining 10 μg (per lane)of various purified 2H7 V_(H) L11S scFv Ig constructs of the invention,indicating an apparent molecular weight for each construct in referenceto a standard molecular weight marker in lane 1. See Example 41.

FIG. 66 compares the CH2 domain sequences of four different human IgGregions, hIgG1, hIgG2, hIgG3, hIgG4, hIgG4, and one rat region, rIgG2b(SEQ ID NOs:694-698). Point mutations affecting ADCC and CDC are labeledwith arrows. See Example 52.

FIG. 67 demonstrates the ability of various 2H7 V_(H) L11S scFv Igconstructs to mediate ADCC in CHO and Lec13 CHO transiently transfectedcells, indicating that constructs expressed in Lec 13 CHO cells had a20% increase in specific killing over the same construct expressed inregular CHO cells. See Example 42.

FIG. 68 shows SDS-PAGE analysis, both reduced and nonreduced, of highand low affinity alleles of soluble CD 16(ED) (SSS-S)H P283S CH2 WCH3(SEQ ID NOs:422 and 426). See Example 43.

FIG. 69 demonstrates the different binding capabilities of the high andlow affinity CD16 fusion proteins to 2H7 V_(H) L11S scFv (CSC-S) WCH2WCH3 or 2H7 V_(H) L11S scFv (SSS-S) WCH2 WCH3, and indicates a loss ofhigh and low affinity allele binding using P238S CH2 constructs. SeeExample 43.

FIG. 70 shows a diagram of (A) an assay used to detect changes in Fcreceptor binding using FITC conjugated CD16 extracellular domain Igfusion protein with a mutated tail, which eliminates self-associationand B) a mammalian display system using cell surface expression ofconstructs. See Example 44.

FIG. 71 shows the induction of apoptosis in Bjab and Ramos cells byvarious mAbs and scFvIg constructs of the invention. See Example 45.

FIG. 72 illustrates the ability of 2H7 scFv (SSS-S) H WCH2 WCH3 and 2H7scFv (SSS-S) H P238CH2 WCH3 constructs to induce apoptosis in Ramoscells that is mediated by activation of caspase 3. The results indicatethat under these conditions, both constructs bind CD20 and inducedapoptosis.

FIG. 73 illustrates the ability of 2H7 scFv (SSS-S) H WCH2 WCH3 and 2H7scFv (SSS-S) H P238CH2 WCH3 constructs to mediate CDC activity in CD20positive Bjab target cells. The results indicate both constructs had theability to mediate CDC.

FIG. 74 compares the ability of 2H7 scFv (SSS-S) H WCH2 WCH3 and 2H7scFv (SSS-S) H P238CH2 WCH3 constructs to mediate ADCC in Bjab targetcells. The results indicate the 2H7 scFv (SSS-S) H WCH2 WCH3 constructwas very effective and induced high levels of specific killing, whilethe of 2H7 scFv (SSS-S) H P238CH2 WCH3 construct was not effective inmediating ADCC.

FIG. 75 compares the ability of 2H7 scFv (SSS-S) H WCH2 WCH3 and 2H7scFv (SSS-S) H P238CH2 WCH3 constructs to bind soluble CD16 (highaffinity and low affinity forms) in CD20 positive CHO cells. Resultsindicate that 2H7 scFv (SSS-S) H WCH2 WCH3 was able to bind CD16 (bothforms), while 2H7 scFv (SSS-S) H P238CH2 WCH3 was not able to bind CD16(either form).

FIG. 76 compares the ability of 2H7 scFv (SSS-S) H WCH2 WCH3 and 2H7scFv (SSS-S) H P238CH2 WCH3 constructs to bind CD64 positive U937 cells.The results indicate that both constructs had high affinity for FcγRI,and that the P238S mutation selectively reduced binding to FcγRIII.

FIG. 77 illustrates an in vivo experiment in macaques to measure theeffect of where 2H7 scFv (SSS-S) H WCH2 WCH3 and 2H7 scFv (SSS-S) HP238CH2 WCH3 constructs on B cell depletion. The constructs wereadministered one week apart and B cells were measures on days −7, 0, 1,3, 7, 8, 10, 14, 28 and 43 using complete blood counts and two-colorflow cytometry. The results indicate that the 2H7 scFv (SSS-S) H WCH2WCH3 construct resulted in rapid and complete depletion of B cells,which lasted up to 28 days after the second injection. Conversely the2H7 scFv (SSS-S) H P238CH2 WCH3 construct resulted in a slow reductionin B cells to about 50% in the first 2 weeks; however, B cells rapidlybegan to return to regular level shortly after this. This suggests thatADCC mediated by CD16 interaction is likely necessary for rapid andsustained B cell depletion.

FIG. 78 illustrates the SEC profiles of G28-1 VL11S scFv (SSS) H WCH2WCH3 and G28-1 VL11S scFv (SSC) H WCH2 WCH3 constructs. The constructwith an SSS hinge generated a single uniform peak at approximately75-100 Kd, while the construct with a SSC hinge generated a smaller formand other heterogeneous forms, including one at greater than 200 Kd.

FIG. 79 illustrates the binding ability of G28-1 VL11S scFv (SSS) H WCH2WCH3 and G28-1 VL11S scFv (SSC) H WCH2 WCH3 constructs in B celllymphoma cells: Bjab, Ramos, WIL-2, Namalwa and Raji. The results in (a)indicate that G28-1 VL11S scFv (SSS) H WCH2 WCH3 bound to Bjab and Ramoscells, and moderately to WIL-2 cells, and at lower levels to Namalwa andRaji cells. The results in (b) indicate the G28-1 VL11S scFv (SSC) HWCH2 WCH3 bound to Bjab cells, and moderately to and Ramos and WIL-2cells, and at lower levels to Namalwa and Raji cells.

FIG. 80 illustrates annexin and v-propidium iodide binding in Ramoscells incubated with G28-1 VL11S scFv (SSS) H WCH2 WCH3 and G28-1 VL11SscFv (SSC) H WCH2 WCH3 constructs overnight. The results indicate thatboth constructs induced apoptosis; however, the construct with the (SSC)hinge induced more apoptosis than the construct with the (SSS) hinge.

FIG. 81 illustrates the ability of G28-1 VL11S scFv (SSS) H WCH2 WCH3and G28-1 VL11S scFv (SSC) H WCH2 WCH3 constructs to inhibitproliferation of Ramos cells. The results indicate that both constructsinhibited proliferation.

FIG. 82 illustrates the ability of G28-1 VL11S scFv (SSS) H WCH2 WCH3and G28-1 VL11S scFv (SSC) H WCH2 WCH3 and 2H7 scFv (CSS) H WCH2 WCH3constructs to induce apoptosis in Ramos B cells, alone and in differentcombinations. The results shown in this experiment indicate that theboth G28-1 constructs were more efficient than the 2H7 construct. Theresults also indicate that the G28-1 VL11S scFv (SSS) H WCH2 WCH3construct was more efficient than the G28-1 VL11S scFv (SSC) H WCH2 WCH3construct. However, the amount of apoptosis was greatest when the 2H7and the G28-1 constructs were used in combination.

FIG. 83 compares the ability of G28-1 VL11S scFv (SSS) H WCH2 WCH3,G28-1 VL11S scFv (SSC) H WCH2 WCH3 and 2H7 scFv (CSS) H WCH2 WCH3constructs to mediate CDC in Ramos B cells. The results indicate thatall the constructs mediated CDC. The 2H7 construct was the mostefficient, followed by G28-1 VL11S scFv (SSC) H WCH2 WCH3, then G28-1VL11S scFv (SSS) H WCH2 WCH3.

FIG. 84 compares the ability of the G28-1 VL11S scFv (SSS) H WCH2 WCH3,G28-1 VL11S scFv (SSC) H WCH2 WCH3 and 2H7 scFv (CSS) H WCH2 WCH3constructs to mediate ADCC in Ramos B cells. The results indicate thatthe G28-1 VL11S scFv (SSS) H WCH2 WCH3 and G28-1 VL11S scFv (SSC) H WCH2WCH3 constructs were able to mediate ADCC, while the 2H7 scFv (CSS) HWCH2 WCH3 construct ability to mediate ADCC was lower, but still higherthan the level of natural killing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel molecules useful, forexample, as therapeutics, as well as for other purposes includingdiagnostic and research purposes. Such molecules have, for example,antigen binding or other binding function(s) and, for example, one ormore effector functions. The invention includes molecular constructs,including binding domain-immunoglobulin fusion proteins, and relatedcompositions and methods, which will be useful in immunotherapeutic andimmunodiagnostic applications, and in research methods, and which offercertain advantages over antigen-specific compounds and polypeptides ofthe prior art. The constructs, including fusion proteins, of the presentinvention are preferably single polypeptide chains that comprise, inpertinent part, the following fused or otherwise connected domains orregions: a binding region construct, such as a binding domain orpolypeptide, a connecting region construct including, for example, anative or engineered immunoglobulin hinge region polypeptide, and a tailregion construct, including, for example, a construct that may comprise,consist essentially of, or consist of, a native or engineeredimmunoglobulin heavy chain CH2 constant region polypeptide and a nativeor engineered immunoglobulin heavy chain CH3 constant regionpolypeptide. According to certain embodiments that are particularlyuseful for gene therapy, the constructs, including fusion proteins, ofthe present invention may further comprise a native or engineered plasmamembrane anchor domain. According to certain other preferred embodimentsthe constructs, including fusion proteins, of the present invention mayfurther include a tail region having a native or engineeredimmunoglobulin heavy chain CH4 constant region polypeptide. Inparticularly preferred embodiments, the binding regions, such aspolypeptide domains, of which the constructs, including bindingdomain-immunoglobulin fusion proteins, are comprised are, or are derivedfrom, polypeptides that are the products of human gene sequences, butthe invention need not be so limited and may in fact relate toconstructs, including binding domain-immunoglobulin fusion proteins, asprovided herein that are derived from any natural or artificial source,including genetically engineered and/or mutated polypeptides.

The present invention relates in part to the surprising observation thatthe novel constructs, including binding domain-immunoglobulin fusionproteins, described herein are capable of immunological activity. Morespecifically, these proteins retain the ability to participate in wellknown immunological effector activities including, for example, antibodydependent cell mediated cytotoxicity (e.g., subsequent to antigenbinding on a cell surface, engagement and induction of cytotoxiceffector cells bearing appropriate Fc receptors, such as Natural Killercells bearing FcRγIII, under appropriate conditions) and/or complementfixation in complement dependent cytotoxicity (e.g., subsequent toantigen binding on a cell surface, recruitment and activation ofcytolytic proteins that are components of the blood complement cascade)despite having structures not be expected to be capable of promotingsuch effector activities or to promtion of such activities as describedherein. For reviews of ADCC and CDC see, e.g., Carter, 2001 Nat. Rev.Canc. 1:118; Sulica et al., 2001 Int. Rev. Immunol. 20:371; Maloney etal., 2002 Semin. Oncol. 29:2; Sondel et al., 2001 Hematol Oncol ClinNorth Am 15(4):703-21; Maloney 2001 Anticanc. Drugs 12 Suppl.2:1-4. IgAactivation of complement by the alternative pathway is described, forexample, in Schneiderman et al., 1990 J. Immunol. 145:233. As describedin greater detail below, ADCC, complement fixation, and CDC areunexpected functions for constructs, including fusion proteins,comprising for example immunoglobulin heavy chain regions and having thestructures described herein, and in particular for immunoglobulin fusionproteins comprising, for example, immunoglobulin hinge regionpolypeptides that are compromised in their ability to form interchain,homodimeric disulfide bonds.

Another advantage afforded by the present invention is constructs,including binding domain-immunoglobulin fusion polypeptides, of theinvention that can be produced in substantial quantities that aretypically greater than those routinely attained with single-chainantibody constructs of the prior art, for example. In preferredembodiments, constructs, including the binding domain-immunoglobulinfusion polypeptides, of the present invention are recombinantlyexpressed in mammalian or other desired and useful expression systems,which offer the advantage of providing polypeptides that are stable invivo (e.g., under physiological conditions). According to non-limitingtheory, such stability may derive in part from posttranslationalmodifications, and specifically glycosylation. Production of theconstructs, including binding domain-immunoglobulin fusion proteinconstructs, of the invention via recombinant mammalian expression hasbeen attained in static cell cultures at a level of greater than 50 mgprotein per liter culture supernatant and has been routinely observed insuch cultures at 10-50 mg/liter, such that preferably at least 10-50mg/liter may be produced under static culture conditions; alsocontemplated are enhanced production, in whole or in part, of theprotein constructs of the invention using art-accepted scale-upmethodologies such as “fed batch” (i.e., non-static) production, whereyields of at least 5-500 mg/l, and in some instances at least 0.5-1gm/l, depending on the particular protein product, are obtained.

A construct, including a binding domain polypeptide, according to thepresent invention may be, for example, any polypeptide that possessesthe ability to specifically recognize and bind to a cognate biologicalmolecule or complex of more than one molecule or assembly or aggregate,whether stable or transient, of such a molecule. Such molecules includeproteins, polypeptides, peptides, amino acids, or derivatives thereof;lipids, fatty acids or the like, or derivatives thereof; carbohydrates,saccharides or the like or derivatives thereof; nucleic acids,nucleotides, nucleosides, purines, pyrimidines or related molecules, orderivatives thereof, or the like; or any combination thereof such as,for example, glycoproteins, glycopeptides, glycolipids, lipoproteins,proteolipids; or any other biological molecule that may be present in abiological sample. Biological samples may be provided, for example, byobtaining a blood sample, biopsy specimen, tissue explant, organculture, biological fluid or any other tissue or cell or otherpreparation from a subject or a biological source. The subject orbiological source may, for example, be a human or non-human animal, aprimary cell culture or culture adapted cell line including but notlimited to genetically engineered cell lines that may containchromosomally integrated or episomal recombinant nucleic acid sequences,immortalized or immortalizable cell lines, somatic cell hybrid celllines, differentiated or differentiatable cell lines, transformed celllines and the like, etc. In certain preferred embodiments of theinvention, the subject or biological source may be suspected of havingor being at risk for having a disease, disorder or condition, includinga malignant disease, disorder or condition or a B cell disorder, whichin certain further embodiments may be an autoimmune disease, and incertain other embodiments of the invention the subject or biologicalsource may be known to be free of a risk or presence of such disease,disorder or condition.

A binding region, including a binding domain polypeptide, for example,may be any naturally occurring, synthetic, semi-synthetic, and/orrecombinantly produced binding partner for a biological or othermolecule that is a target structure of interest, herein sometimesreferred to as an “antigen” but intended according to the presentdisclosure to encompass any target biological or other molecule to whichit is desirable to have the subject invention, for example, a fusionprotein, bind or specifically bind. Constructs of the invention,including binding domain-immunoglobulin fusion proteins, are defined tobe “immunospecific” or capable of binding to a desired degree, includingspecifically binding, if they bind a desired target molecule such as anantigen as provided herein, at a desired level, for example, with aK_(a) of greater than or equal to about 10⁴ M⁻¹, preferably of greaterthan or equal to about 10⁵ M⁻¹, more preferably of greater than or equalto about 10⁶ M⁻¹ and still more preferably of greater than or equal toabout 10⁷ M⁻¹. Affinities of even greater than about 10⁷ M⁻¹ are stillmore preferred, such as affinities equal to or greater than about 10⁷M⁻¹, about 10⁸ M⁻¹, and about 10⁹ M⁻¹, and about 10¹⁰ M⁻¹. Affinities ofbinding domain-immunoglobulin fusion proteins according to the presentinvention can be readily determined using conventional techniques, forexample those described by Scatchard et al., 1949 Ann. N.Y. Acad. Sci.51:660. Such determination of fusion protein binding to target antigensof interest can also be performed using any of a number of known methodsfor identifying and obtaining proteins that specifically interact withother proteins or polypeptides, for example, a yeast two-hybridscreening system such as that described in U.S. Pat. No. 5,283,173 andU.S. Pat. No. 5,468,614, or the equivalent.

Preferred embodiments of the subject invention constructs, for example,binding domain-immunoglobulin fusion proteins, comprise binding regionsor binding domains that may include, for example, at least one native orengineered immunoglobulin variable region polypeptide, such as all or aportion or fragment of a native or engineered heavy chain and/or anative or engineered light chain V-region, provided it is capable ofbinding or specifically binding an antigen or other desired targetstructure of interest at a desired level of binding and selectivity. Inother preferred embodiments the binding region or binding domaincomprises, consists essentially of, or consists of, a single chainimmunoglobulin-derived Fv product, for example, and scFv, which mayinclude all or a portion of at least one native or engineeredimmunoglobulin light chain V-region and all or a portion of at least onenative or engineered immunoglobulin heavy chain V-region, and a linkerfused or otherwise connected to the V-regions; preparation and testingsuch constructs are described in greater detail herein. Otherpreparation and testing methods are well known in the art.

As described herein and known in the art, immunoglobulins compriseproducts of a gene family the members of which exhibit a high degree ofsequence conservation. Amino acid sequences of two or moreimmunoglobulins or immunoglobulin domains or regions or portions thereof(e.g., V_(H) domains, V_(L) domains, hinge regions, CH2 constantregions, CH3 constant regions) may be aligned and analyzed. Portions ofsequences that correspond to one another may be identified, forinstance, by sequence homology. Determination of sequence homology maybe determined with any of a number of sequence alignment and analysistools, including computer algorithms well known to those of ordinaryskill in the art, such as Align or the BLAST algorithm (Altschul, 1991J. Mol. Biol. 219:555-565; Henikoff and Henikoff, 1992 Proc. Natl. Acad.Sci. USA 89:10915-10919), which is available at the NCBI website(http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST). Default parameters may beused.

Portions, for example, of a particular immunoglobulin reference sequenceand of any one or more additional immunoglobulin sequences of interestthat may be compared to a reference sequence. “Corresponding” sequences,regions, fragments or the like, may be identified based on theconvention for numbering immunoglobulin amino acid positions accordingto Kabat, Sequences of Proteins of Immunological Interest, (5^(th) ed.Bethesda, Md.: Public Health Service, National Institutes of Health(1991)). For example, according to this convention, the immunoglobulinfamily to which an immunoglobulin sequence of interest belongs isdetermined based on conservation of variable region polypeptide sequenceinvariant amino acid residues, to identify a particular numbering systemfor the immunoglobulin family, and the sequence(s) of interest can thenbe aligned to assign sequence position numbers to the individual aminoacids which comprise such sequence(s). Preferably at least about 70%,more preferably at least about 80%-85% or about 86%-89%, and still morepreferably at least about 90%, about 92%, about 94%, about 96%, about98% or about 99% of the amino acids in a given amino acid sequence of atleast about 1000, more preferably about 700-950, more preferably about350-700, still more preferably about 100-350, still more preferablyabout 80-100, about 70-80, about 60-70, about 50-60, about 40-50 orabout 30-40 consecutive amino acids of a sequence, are identical to theamino acids located at corresponding positions in a reference sequencesuch as those disclosed by Kabat (1991) or in a similar compendium ofrelated immunoglobulin sequences, such as may be generated from publicdatabases (e.g., Genbank, SwissProt, etc.) using sequence alignmenttools such as, for example, those described above. In certain preferredembodiments, an immunoglobulin sequence of interest or a region,portion, derivative or fragment thereof is greater than about 95%identical to a corresponding reference sequence, and in certainpreferred embodiments such a sequence of interest may differ from acorresponding reference at no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 amino acid positions.

For example, in certain embodiments the present invention is directed toa construct, including a binding domain-immunoglobulin fusion protein,comprising in pertinent part a human or other species immunoglobulinheavy chain variable region polypeptide comprising a mutation,alteration or deletion at an amino acid at a location or locationscorresponding to one or more of amino acid positions 9, 10, 11, 12, 108,110, 111, and 112 in, for example, SEQ ID NO:212, which comprises, forexample, a murine V_(H)-derived sequence. At a relatively limited numberof immunoglobulin V_(H) sequence positions, for example, includingposition 11, amino acid conservation is observed in the overwhelmingmajority of V_(H) sequences analyzed across mammalian species lines(e.g., Leull, Va137, Gly44, Leu45, Trp47; Nguyen et al., 1998 J. Mol.Biol. 275:413). Various such amino acid residues, and hence their sidechains, are located at the surface of the variable domain (V_(H)). Theymay contact residues of the C_(H)1 (e.g., Leu11) and the V_(L) domains(e.g., Va137, Gly44, Leu45, and Trp47) and may, in the absence of lightchains, contribute to stability and solubility of the protein (see,e.g., Chothia et al., 1985 J. Mol. Biol. 186:651; Muyldermans et al.,1994 Prot. Engineer. 7:1129; Desmyter et al., 1996 Nat. Struct. Biol.3:803; Davies et al., 1994 FEES Lett. 339:285). In certain embodiments,for example, the present invention is also directed to a construct,including a binding domain-immunoglobulin fusion protein, comprising inpertinent part a human immunoglobulin light chain variable regionpolypeptide, or an immunoglobulin light chain variable regionpolypeptide from another species, comprising a mutation, alteration ordeletion at an amino acid at a location or locations corresponding toone or more of amino acid positions 12, 80, 81, 82, 83, 105, 106, 107and 108. In still other certain embodiments, for example, the presentinvention is directed to a construct, including a bindingdomain-immunoglobulin fusion protein, comprising in pertinent part (1) ahuman immunoglobulin heavy chain variable region polypeptide, or animmunoglobulin light chain variable region polypeptide from anotherspecies, comprising, consisting essentially of, or consisting of, saidheavy chain sequence having a mutation, alteration or deletion at alocation or locations corresponding to one or more of amino acidpositions 9, 10, 11, 12, 108, 110, 111, and 112, and (2) a humanimmunoglobulin light chain variable region polypeptide, or animmunoglobulin light chain variable region polypeptide from anotherspecies, comprising, consisting essentially of, or consisting of, saidlight chain sequence having a mutation, alteration or deletion at alocation or locations corresponding to one or more of amino acidpositions 12, 80, 81, 82, 83, 105, 106, 107 and 108.

As another example, by reference to immunoglobulin sequence compendiaand databases such as those cited above, for example, the relatedness oftwo or more immunoglobulin sequences to each other can readily andwithout undue experimentation be established in a manner that permitsidentification of the animal species of origin, the class and subclass(e.g., isotype) of a particular immunoglobulin or immunoglobulin regionpolypeptide sequence. Any immunoglobulin variable region polypeptidesequence, including native or engineered V_(H) and/or V_(L) and/orsingle-chain variable region (sFv) sequences or other native orengineered V region-derived sequences or the like, may be used as abinding region or binding domain. Engineered sequences includesimmunoglobulin sequences from any species, preferably human or mouse,for example, that include, for example, a mutation, alteration ordeletion at an amino acid at a location or locations corresponding toone or more of amino acid positions 9, 10, 11, 12, 108, 110, 111, and112 in a heavy chain variable region sequence or an scFv, and/or amutation, alteration or deletion at a location or locationscorresponding to one or more of amino acid positions 12, 80, 81, 82, 83,105, 106, 107 and 108 in a light chain variable region sequence or anscFv.

Various preferred embodiments include, for example, native or engineeredimmunoglobulin V region polypeptide sequences derived, for example, fromantibodies including monoclonal antibodies such as murine or otherrodent antibodies, or antibodies or monoclonal antibodies derived fromother sources such as goat, rabbit, equine, bovine, camelid or otherspecies, including transgenic animals, and also including human orhumanized antibodies or monoclonal antibodies. Non-limiting examplesinclude variable region polypeptide sequences derived from monoclonalantibodies such as those referenced herein and/or described in greaterdetail in the Examples below, for instance, CD20-binding or specificmurine monoclonal antibodies (e.g., 2H7), other CD20-binding or specificmurine monoclonal antibodies that are not 1F5 antibodies, monoclonalantibody L6 (specific for a carbohydrate-defined epitope and availablefrom American Type Culture Collection, Manassas, Va., as hybridomaHB8677), and monoclonal antibodies that bind to or are specific for CD28(e.g., monoclonal antibody 2E12), CD40, CD80, CD137 (e.g., monoclonalantibody 5B9 or monoclonal antibody 1D8 which recognizes the murinehomologue of CD137, 41BB) and CD152 (CTLA-4).

Other binding regions, including binding domain polypeptides, maycomprise any protein or portion thereof that retains the ability to bindor specifically bind to an antigen as provided herein, includingnon-immunoglobulins. Accordingly the invention contemplates constructs,including fusion proteins, comprising binding region or binding domainpolypeptides that are derived from polypeptide ligands such as hormones,cytokines, chemokines, and the like; cell surface or soluble receptorsfor such polypeptide ligands; lectins; intercellular adhesion receptorssuch as specific leukocyte integrins, selectins, immunoglobulin genesuperfamily members, intercellular adhesion molecules (ICAM-1, -2, -3)and the like; histocompatibility antigens; etc.

Examples of cell surface receptors useful in the preparation of, or as,binding regions, or that may provide a binding domain polypeptide, andthat may also be selected as a target molecule or antigen to which aconstruct, including for example, a binding domain-Ig fusion protein ofthe present invention desirably binds, include the following, or thelike: HER1 (e.g., GenBank Accession Nos. U48722, SEG_HEGFREXS, KO03193),HER2 (Yoshino et al., 1994 J. Immunol. 152:2393; Disis et al., 1994Canc. Res. 54:16; see also, e.g., GenBank Acc. Nos. X03363, M17730,SEG_HUMHER20), HER3 (e.g., GenBank Acc. Nos. U29339, M34309), HER4(Plowman et al., 1993 Nature 366:473; see also e.g., GenBank Acc. Nos.L07868, T64105), epidermal growth factor receptor (EGFR) (e.g., GenBankAcc. Nos. U48722, SEG_HEGFREXS, K03193), vascular endothelial cellgrowth factor (e.g., GenBank No. M32977), vascular endothelial cellgrowth factor receptor (VEGF) (e.g., GenBank Acc. Nos. AF022375,1680143, U48801, X62568), insulin-like growth factor-I (e.g., GenBankAcc. Nos. X00173, X56774, X56773, X06043, see also European Patent No.GB 2241703), insulin-like growth factor-II (e.g., GenBank Acc. Nos.X03562, X00910, SEG_HUMGFIA, SEG_HUMGFI2, M17863, M17862), transferrinreceptor (Trowbridge and Omary, 1981 Proc. Nat. Acad. USA 78:3039; seealso e.g., GenBank Acc. Nos. X01060, M11507), estrogen receptor (e.g.,GenBank Acc. Nos. M38651, X03635, X99101, U47678, M12674), progesteronereceptor (e.g., GenBank Acc. Nos. X51730, X69068, M15716), folliclestimulating hormone receptor (FSH-R) (e.g., GenBank Acc. Nos. Z34260,M65085), retinoic acid receptor (e.g., GenBank Acc. Nos. L12060, M60909,X77664, X57280, X07282, X06538), MUC-1 (Barnes et al., 1989 Proc. Nat.Acad. Sci. USA 86:7159; see also e.g., GenBank Acc. Nos. SEG_MUSMUCIO,M65132, M64928) NY-ESO-1 (e.g., GenBank Acc. Nos. AJ003149, U87459), NA17-A (e.g., European Patent No. WO 96/40039), Melan-A/MART-1 (Kawakamiet al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBankAcc. Nos. U06654, U06452), tyrosinase (Topalian et al., 1994 Proc. Nat.Acad. Sci. USA 91:9461; see also e.g., GenBank Acc. Nos. M26729,SEG_HUMTYR0, see also Weber et al., J. Clin. Invest (1998) 102:1258),Gp-100 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; seealso e.g., GenBank Acc. No. S73003, see also European Patent No. EP668350; Adema et al., 1994 J. Biol. Chem. 269:20126), MAGE (van denBruggen et al., 1991 Science 254:1643; see also e.g, GenBank Acc. Nos.U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693,U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340,U10339, L18920, U03735, M77481), BAGE (e.g., GenBank Acc. No. U19180;see also U.S. Pat. Nos. 5,683,886 and 5,571,711), GAGE (e.g., GenBankAcc. Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144,U19143, U19142), any of the CTA class of receptors including inparticular HOM-MEL-40 antigen encoded by the SSX2 gene (e.g., GenBankAcc. Nos. X86175, U90842, U90841, X86174), carcinoembyonic antigen (CEA,Gold and Freedman, 1985 J. Exp. Med. 121:439; see also e.g., GenBankAcc. Nos. SEG_HUMCEA, M59710, M59255, M29540), and PyLT (e.g., GenBankAcc. Nos. J02289, J02038).

Additional cell surface receptors that may be sources of binding regionor binding domain polypeptides or portions thereof, or that may betargets, including target antigens, include the following, or the like:CD2 (e.g., GenBank Acc. Nos. Y00023, SEG_HUMCD2, M16336, M16445,SEG_MUSCD2, M14362), 4-1BB (CDw137, Kwon et al., 1989 Proc. Nat. Acad.Sci. USA 86:1963, 4-1BB ligand (Goodwin et al., 1993 Eur. J. Immunol.23:2361; Melero et al., 1998 Eur. J. Immunol. 3:116), CD5 (e.g., GenBankAcc. Nos. X78985, X89405), CD10 (e.g., GenBank Acc. Nos. M81591, X76732)CD27 (e.g., GenBank Acc. Nos. M63928, L24495, L08096), CD28 (June etal., 1990 Immunol. Today 11:211; see also, e.g., GenBank Acc. Nos.J02988, SEG_HUMCD28, M34563), CD152/CTLA-4 (e.g., GenBank Acc. Nos.L15006, X05719, SEG_HUMIGCTL), CD40 (e.g., GenBank Acc. Nos. M83312,SEG_MUSC040A0, Y10507, X67878, X96710, U15637, L07414), interferon-γ(IFN-γ; see, e.g., Farrar et al. 1993 Ann. Rev. Immunol. 11:571 andreferences cited therein, Gray et al. 1982 Nature 295:503, Rinderknechtet al. 1984 J. Biol. Chem. 259:6790, DeGrado et al. 1982 Nature300:379), interleukin-4 (IL-4; see, e.g., 53^(rd) Forum in Immunology,1993 Research in Immunol. 144:553-643; Banchereau et al., 1994 in TheCytokine Handbook, 2^(nd) ed., A. Thomson, ed., Academic Press, NY, p.99; Keegan et al., 1994 J. Leukocyt. Biol. 55:272, and references citedtherein), interleukin-17 (IL-17) (e.g., GenBank Acc. Nos. U32659,U43088) and interleukin-17 receptor (IL-17R) (e.g., GenBank Acc. Nos.U31993, U58917). Notwithstanding the foregoing, the present inventionexpressly does not encompass any immunoglobulin fusion protein that isdisclosed in U.S. Pat. No. 5,807,734, or U.S. Pat. No. 5,795,572.

Additional cell surface receptors that may be sources of binding regionor binding domain polypeptides or portions thereof, or that may serve astargets including target antigens or binding sites include thefollowing, or the like: CD59 (e.g., GenBank Acc. Nos. SEG_HUMCD590,M95708, M34671), CD48 (e.g., GenBank Acc. Nos. M59904), CD58/LFA-3(e.g., GenBank Acc. No. A25933, Y00636, E12817; see also JP1997075090-A), CD72 (e.g., GenBank Acc. Nos. AA311036, 540777, L35772),CD70 (e.g., GenBank Acc. Nos. Y13636, S69339), CD80/B7.1 (Freeman etal., 1989 J. Immunol. 43:2714; Freeman et al., 1991 J. Exp. Med.174:625; see also e.g., GenBank Acc. Nos. U33208, 1683379), CD86/B7.2(Freeman et al., 1993 J. Exp. Med. 178:2185, Boriello et al., 1995 J.Immunol. 155:5490; see also, e.g., GenBank Acc. Nos. AF099105,SEG_MMB72G, U39466, U04343, SEG_HSB725, L25606, L25259), B7-H1/B7-DC(e.g., Genbank Acc. Nos. NM_(—)014143, AF177937, AF317088; Dong et al.,2002 Nat. Med. June 24 [epub ahead of print], PMID 12091876; Tseng etal., 2001 J. Exp. Med. 193:839; Tamura et al., 2001 Blood 97:1809; Donget al., 1999 Nat. Med. 5:1365), CD40 ligand (e.g., GenBank Acc. Nos.SEG_HUMCD40L, X67878, X65453, L07414), IL-17 (e.g., GenBank Acc. Nos.U32659, U43088), CD43 (e.g., GenBank Acc. Nos. X52075, J04536), ICOS(e.g., Genbank Acc. No. AH011568), CD3 (e.g., Genbank Acc. Nos.NM_(—)000073 (gamma subunit), NM_(—)000733 (epsilon subunit), X73617(delta subunit)), CD4 (e.g., Genbank Acc. No. NM_(—)000616), CD25 (e.g.,Genbank Acc. No. NM_(—)000417), CD8 (e.g., Genbank Acc. No.M12828),CD11b (e.g., Genbank Acc. No. J03925), CD14 (e.g., Genbank Acc. No.XM_(—)039364), CD56 (e.g., Genbank Acc. No.U63041), CD69 (e.g., GenbankAcc. No.NM_(—)001781) and VLA-4 (α₄β₇) (e.g., GenBank Acc. Nos. L12002,X16983, L20788, U97031, L24913, M68892, M95632). The following cellsurface receptors are typically associated with B cells: CD19 (e.g.,GenBank Acc. Nos. SEG_HUMCD19W0, M84371, SEG_MUSCD19W, M62542), CD20(e.g., GenBank Acc. Nos. SEG_HUMCD20, M62541), CD22 (e.g., GenBank Acc.Nos. 1680629, Y10210, X59350, U62631, X52782, L16928), CD30 (e.g.,Genbank Acc. Nos. M83554, D86042), CD153 (CD30 ligand, e.g., GenBankAcc. Nos. L09753, M83554), CD37 (e.g., GenBank Acc. Nos. SEG_MMCD37X,X14046, X53517), CD50 (ICAM-3, e.g., GenBank Acc. No. NM_(—)002162),CD106 (VCAM-1) (e.g., GenBank Acc. Nos. X53051, X67783, SEG_MMVCAM1C,see also U.S. Pat. No. 5,596,090), CD54 (ICAM-1) (e.g., GenBank Acc.Nos. X84737, 582847, X06990, J03132, SEG_MUSICAMO), interleukin-12 (see,e.g., Reiter et al, 1993 Crit. Rev. Immunol. 13:1, and references citedtherein), CD134 (OX40, e.g., GenBank Acc. No. AJ277151), CD137 (41BB,e.g., GenBank Acc. No. L12964, NM_(—)001561), CD83 (e.g., GenBank Acc.Nos. AF001036, AL021918), DEC-205 (e.g., GenBank Acc. Nos. AF011333,U19271).

Constructs, including binding domain-immunoglobulin fusion proteins, ofthe present invention comprise, for example, a binding domain, such as abinding domain polypeptide that, according to certain particularlypreferred embodiments, is capable of binding or specifically binding atleast one target, for example, a target antigen or other binding sitethat is present on an immune effector cell. According to non-limitingtheory, such constructs, including for example bindingdomain-immunoglobulin fusion proteins, may advantageously recruitdesired immune effector cell function(s) in a therapeutic context, whereit is well known that immune effector cells having different specializedimmune functions can be identified or distinguished from one another onthe basis of their differential expression of a wide variety of cellsurface antigens, including many of the antigens described herein towhich constructs of the invention including binding domain polypeptidescan specifically bind. As noted herein, immune effector cells includeany cell that is capable of directly mediating an activity that is acomponent of immune system function, including cells having suchcapability naturally or as a result of genetic engineering.

In certain embodiments an immune effector cell comprises a cell surfacereceptor for an immunoglobulin or other peptide binding molecule, suchas a receptor for an immunoglobulin constant region and including theclass of receptors commonly referred to as “Fc receptors” (“FcR”s). Anumber of FcRs have been structurally and/or functionally characterizedand are well known in the art, including FcR having specific abilitiesto interact with a restricted subset of immunoglobulin heavy chainisotypes, or that interact with Fc domains with varying affinities,and/or which may be expressed on restricted subsets of immune effectorcells under certain conditions (e.g., Kijimoto-Ochichai et al., 2002Cell Mol. Life. Sci. 59:648; Davis et al., 2002 Curr. Top. Microbiol.Immunol. 266:85; Pawankar, 2001 Curr. Opin. Allerg. Clin. Immunol. 1:3;Radaev et al., 2002 Mol. Immunol. 38:1073; Wurzburg et al., 2002 Mol.Immunol. 38:1063; Sulica et al., 2001 Int. Rev. Immunol. 20:371;Underhill et al., 2002 Ann. Rev. Immunol. 20:825; Coggeshall, 2002 Curr.Dir. Autoimm. 5:1; Mimura et al., 2001 Adv. Exp. Med. Biol. 495:49;Baumann et al., 2001 Adv. Exp. Med. Biol. 495:219; Santoso et al., 2001Ital. Heart J. 2:811; Novak et al., 2001 Curr. Opin. Immunol. 13:721;Fossati et al., 2001 Eur. J. Clin. Invest. 31:821).

Cells that are capable of mediating ADCC are preferred examples ofimmune effector cells according to the present invention. Otherpreferred examples include Natural Killer cells, tumor-infiltrating Tlymphocytes (TILs), cytotoxic T lymphocytes, and granulocytic cells suchas cells that comprise allergic response mechanisms. Immune effectorcells thus include, but are not limited to, cells of hematopoieticorigins including cells at various stages of differentiation withinmyeloid and lymphoid lineages and which may (but need not) express oneor more types of functional cell surface FcR, such as T lymphocytes, Blymphocytes, NK cells, monocytes, macrophages, dendritic cells,neutrophils, basophils, eosinophils, mast cells, platelets,erythrocytes, and precursors, progenitors (e.g., hematopoietic stemcells), as well as quiescent, activated, and mature forms of such cells.Other immune effector cells may include cells of non-hematopoieticorigin that are capable of mediating immune functions, for example,endothelial cells, keratinocytes, fibroblasts, osteoclasts, epithelialcells, and other cells. Immune effector cells may also include cellsthat mediate cytotoxic or cytostatic events, or endocytic, phagocytic,or pinocytotic events, or that effect induction of apoptosis, or thateffect microbial immunity or neutralization of microbial infection, orcells that mediate allergic, inflammatory, hypersensitivity and/orautoimmune reactions.

Allergic response mechanisms are well known in the art and include anantigen (e.g., allergen)-specific component such as an immunoglobulin(e.g., IgE), as well as the cells and mediators which comprise sequelaeto allergen-immunoglobulin (e.g., IgE) encounters (e.g., Ott et al.,2000 J. Allerg. Clin. Immunol. 106:429; Barnes, 2000 J. Allerg. Clin.Immunol. 106:5; Togias, 2000 J. Allerg. Clin. Immunol. 105:S599; Akdiset al., 2000 Int. Arch. Allerg. Immunol. 121:261; Beach, 2000 Occup.Med. 15:455). Particularly with regard to constructs, including bindingdomain-immunoglobulin fusion proteins, of the present invention thatinteract with FcR, certain embodiments of the present inventioncontemplate constructs including fusion proteins that comprise one ormore IgE-derived domains including, for example, those that are capableof inducing an allergic response mechanism that comprises IgE-specificFcR, or portions thereof, which IgE-specific FcRs include those notedabove and described or identified in the cited articles. Without wishingto be bound by particular theory or mechanism, and as disclosed herein,constructs, including fusion proteins, of the present invention maycomprise portions of IgE heavy chain Fc domain polypeptides, forexample, native or engineered IgE CH3 and CH4 domains, whether providedor expressed as cell surface proteins (e.g., with a plasma membraneanchor domain) or as soluble or otherwise not cell-bound proteins (e.g.,without a plasma membrane anchor domain). Further according tonon-limiting theory, recruitment and induction of an allergic responsemechanism (e.g., an FcR-epsilon expressing immune effector cell) mayproceed as the result of either or both of the presence of an IgE Fcdomain or portion thereof as described herein (e.g., one that is capableof triggering an allergic mechanism by FcR crosslinking) and thepresence of a target such as a antigen to which the binding region, forexample a binding domain, binds or specifically binds. The presentinvention therefore exploits induction of allergic response mechanismsin heretofore unappreciated contexts, such as treatment of a malignantcondition or a B cell disorder, including those described or referencedherein.

An immunoglobulin hinge region polypeptide includes any hinge peptide orpolypeptide that occurs naturally, as an artificial peptide or as theresult of genetic engineering and that is situated, for example, in animmunoglobulin heavy chain polypeptide between the amino acid residuesresponsible for forming intrachain immunoglobulin-domain disulfide bondsin CH1 and CH2 regions. Hinge region polypeptides for use in the presentinvention may also include a mutated or otherwise alterd hinge regionpolypeptide. Accordingly, for example, an immunoglobulin hinge regionpolypeptide may be derived from, or may be a portion or fragment of(i.e., one or more amino acids in peptide linkage, typically about15-115 amino acids, preferably about 95-110, about 80-94, about 60-80,or about 5-65 amino acids, preferably about 10-50, more preferably about15-35, still more preferably about 18-32, still more preferably about20-30, still more preferably about 21, 22, 23, 24, 25, 26, 27, 28 or 29amino acids) an immunoglobulin polypeptide chain region classicallyregarded as having hinge function, including those described herein, buta hinge region polypeptide for use in the instant invention need not beso restricted and may include one or more amino acids situated(according to structural criteria for assigning a particular residue toa particular domain that may vary, as known in the art) in an adjoiningimmunoglobulin domain such as a CH1 domain and/or a CH2 domain in thecases of IgG, IgA and IgD (or in an adjoining immunoglobulin domain suchas a CH1 domain and/or a CH3 domain in the case of IgE), or in the caseof certain artificially engineered immunoglobulin constructs, animmunoglobulin variable region domain.

Wild-type immunoglobulin hinge region polypeptides include any known orlater-discovered naturally occurring hinge region that is locatedbetween the constant region domains, CH1 and CH2, of an immunoglobulin,for example, a human immunoglobulin (or between the CH1 and CH3 regionsof certain types of immunoglobulins, such as IgE). For use inconstructing one type of connecting region, the wild-type immunoglobulinhinge region polypeptide is preferably a human immunoglobulin hingeregion polypeptide, preferably comprising a hinge region from a humanIgG, IgA, or IgD immunoglobulin (or the CH2 region of an IgEimmunoglobulin), and more preferably, for example, a hinge regionpolypeptide from a wild-type or mutated human IgG1 isotype as describedherein.

As is known to the art, despite the tremendous overall diversity inimmunoglobulin amino acid sequences, immunoglobulin primary structureexhibits a high degree of sequence conservation in particular portionsof immunoglobulin polypeptide chains, notably with regard to theoccurrence of cysteine residues which, by virtue of their sulfyhydrylgroups, offer the potential for disulfide bond formation with otheravailable sulfydryl groups. Accordingly, in the context of the presentinvention wild-type immunoglobulin hinge region polypeptides for use asconnecting regions include those that feature one or more highlyconserved (e.g., prevalent in a population in a statisticallysignificant manner) cysteine residues, and in certain preferredembodiments a connecting region may comprise, or consist essentially of,or consist of, a mutated hinge region polypeptide may be selected thatcontains less than the number of naturally-occurring cysteines, forexample, zero or one or two cysteine residue(s) in the case of IgG1 andIgG4 hinge regions, and that is derived or constructed from (or using)such a wild-type hinge region sequence.

In certain preferred embodiments wherein the connecting region is ahinge region polypeptide and the hinge region polypeptide is a mutated,engineered or otherwise altered human IgG1 immunoglobulin hinge regionpolypeptide that is derived or constructed from (or using) a wild-typehinge region sequence, it is noted that the wild-type human IgG1 hingeregion polypeptide sequence comprises three non-adjacent cysteineresidues, referred to as a first cysteine of the wild-type hinge region,a second cysteine of the wild-type hinge region and a third cysteine ofthe wild-type hinge region, respectively, proceeding along the hingeregion sequence from the polypeptide N-terminus toward the C-terminus.This can be referred to herein as a “CCC” hinge (or a “WTH”, i.e., awild-type hinge). Examples of mutated or engineered hinge regionsinclude those with no cysteines, which may be referred to herein as an“XXX” hinge (or, for example, as “MH-XXX,” referring to a mutant orengineered hinge with three amino acids or other molecules in place ofnaturally occurring cysteines, such as, for example, “MH-SSS”, whichrefers to a mutant hinge with three serine residues in place of thenaturally occurring cysteine residues. It will be understood that theterm “mutant” refers only to the fact that a different molecule ormolecules is present, or no molecule, at the position of a naturallyoccurring residue and does not refer to any particular method by whichsuch substitution, alteration, or deletion has been carried out.Accordingly, in certain embodiments of the present invention, theconnecting region may be a hinge region polypeptide and the hinge regionpolypeptide is a mutated human IgG1 immunoglobulin hinge regionpolypeptide that contains two cysteine residues and in which the firstcysteine of the wild-type hinge region has not changed or deleted, forexample. This can be referred to as a “MH-CXX” hinge, for example, a“MH-CSC” hinge, in which case the cysteine residue has been replacedwith a serine residue. In certain other embodiments of the presentinvention the mutated human IgG1 immunoglobulin hinge region polypeptidecontains no more than one cysteine residue and include, for example, a“MH-CSS” hinge or a “MH-SSC” hinge or a “MH-CSC” hinge, and in certainother embodiments the mutated human IgG1 immunoglobulin hinge regionpolypeptide contains no cysteine residues such as, for example, a“MH-SSS” hinge.

The constructs, including binding domain-immunoglobulin fusion proteins,of the present invention expressly do not contemplate any fusion proteinthat is disclosed in U.S. Pat. No. 5,892,019. U.S. Pat. No. 5,892,019refers to a human IgG1 hinge region in which the first IgG1 hinge regioncysteine residue has been changed or deleted, but retains both of thesecond and third IgG1 hinge region cysteine residues that correspond tothe second and third cysteines of the wild-type IgG1 hinge regionsequence. The patent states that the first cysteine residue of thewild-type IgG1 hinge region is replaced to prevent interference by thefirst cysteine residue with proper assembly of the polypeptide describedtherein into a dimer. The patent requires that the second and thirdcysteines of the IgG1 hinge region be retained to provide interchaindisulfide linkage between two heavy chain constant regions to promotedimer formation so that the molecule contains has effector function suchas the ability to mediate ADCC.

By contrast and as described herein, the constructs, including thebinding domain-immunogloblin fusion proteins, of the present invention,various of which are capable of ADCC, CDC and/or complement fixation,for example, are not so limited and may comprise, in pertinent part, forexample, (i) a wild-type immunoglobulin hinge region polypeptide, suchas a wild-type human immunoglobulin hinge region polypeptide, forexample, a human IgG1 immunoglobulin hinge region polypeptide, (ii) amutated or otherwise altered immunoglobulin hinge region polypeptide,such as a mutated or otherwise altered human immunoglobulin hinge regionpolypeptide, for example, a mutated or otherwise altered human IgG1immunoglobulin hinge region polypeptide that, for example, is or hasbeen derived or constructed from (or using) a wild-type immunoglobulinhinge region polypeptide or nucleic acid sequence having three or morecysteine residues, wherein the mutated or otherwise altered human IgG1immunoglobulin hinge region polypeptide contains two cysteine residuesand wherein a first cysteine of the wild-type hinge region is notmutated or deleted, (iii) a mutated or otherwise altered immunoglobulinhinge region polypeptide, such as a mutated or otherwise altered humanimmunoglobulin hinge region polypeptide, for example, a mutated orotherwise altered human IgG1 immunoglobulin hinge region polypeptidethat, for example, is or has been derived or constructed from (or using)a wild-type immunoglobulin hinge region polypeptide or nucleic acidsequence having three or more cysteine residues, wherein the mutated orotherwise altered human IgG1 immunoglobulin hinge region polypeptidecontains no more than one cysteine residue, or (iv) a mutated orotherwise altered immunoglobulin hinge region polypeptide, such as amutated or otherwise altered human immunoglobulin hinge regionpolypeptide, for example, a mutated or otherwise altered human IgG1immunoglobulin hinge region polypeptide that is or has been derived orconstructed from (or using) a wild-type immunoglobulin hinge regionpolypeptide or nucleic acid sequence having three or more cysteineresidues, wherein the mutated or otherwise altered (for example, byamino acid change or deletion) human IgG1 immunoglobulin hinge regionpolypeptide contains no cysteine residues. The present invention offersunexpected advantages associated with retention by the constructs,including the fusion proteins, described herein of the ability tomediate ADCC and/or CDC and/or complement fixation notwithstanding thatthe ability to dimerize via IgG1 hinge region interchain disulfide bondsis ablated or compromised by the removal or replacement of one, two orthree hinge region cysteine residues, and even in constructs where thefirst cysteine of an IgG1 hinge region, for example, is not mutated orotherwise altered or deleted.

A connecting region may comprise a mutated or otherwise alteredimmunoglobulin hinge region polypeptide, which itself may comprise ahinge region that has its origin in an immunoglobulin of a species, ofan immunoglobulin isotype or class, or of an immunoglobulin subclassthat is different from that of the tail region, for example, a tailregion comprising, or consisting essentially or, or consisting of, CH2and CH3 domains (or IgE CH3 and CH4 domains). For instance, in certainembodiments of the invention, a construct, for example, a bindingdomain-immunoglobulin fusion protein, may comprise a binding region suchas a binding domain polypeptide that is fused or otherwise connected toan immunoglobulin hinge region polypeptide comprising, or consistingessentially of, or consisting of, a wild-type human IgA hinge regionpolypeptide, or a mutated or otherwise altered human IgA hinge regionpolypeptide that contains zero or only one or more cysteine residues(but less than the wild-type number of cysteines), as described herein,or a wild-type human IgG hinge, such as an IgG1 hinge, regionpolypeptide, or a wild-type human IgE hinge-acting region, i.e., IgE CH2region polypeptide, or a mutated or otherwise altered human IgG hinge,such as an IgG1 hinge, region polypeptide that is or has been mutated orotherwise altered to contain zero, one or two cysteine residues whereinthe first cysteine of the wild-type hinge region is not mutated oraltered or deleted, as also described herein. Such a hinge regionpolypeptide may be fused or otherwise connected to, for example, a tailregion comprising, or consisting essentially of, or consisting of, animmunoglobulin heavy chain CH2 region polypeptide from a different Igisotype or class, for example an IgA or an IgD or an IgG subclass (or aCH3 region from an IgE subclass), which in certain preferred embodimentswill be the IgG1 or IgA or IgE subclass and in certain other preferredembodiments may be any one of the IgG2, IgG3 or IgG4 subclasses.

For example, and as described in greater detail herein, in certainembodiments of the present invention a connecting region may be selectedto be an immunoglobulin hinge region polypeptide, which is or has beenderived from a wild-type human IgA hinge region that naturally comprisesthree cysteines, where the selected hinge region polypeptide istruncated or otherwise altered or substituted relative to the completeand/or naturally-occurring hinge region such that only one or two of thecysteine residues remain (e.g., SEQ ID NOS:35-36). Similarly, in certainother embodiments of the invention, the construct may be bindingdomain-immunoglobulin fusion protein comprising a binding domainpolypeptide that is fused or otherwise connected to an immunoglobulinhinge region polypeptide comprising a mutated or otherwise altered hingeregion polypeptide in which the number of cysteine residues is reducedby amino acid substitution or deletion, for example a mutated orotherwise altered IgG1 hinge region containing zero, one or two cysteineresidues as described herein, or an IgD hinge region containing zerocysteine residues.

A mutated or otherwise altered hinge region polypeptide may thus bederived or constructed from (or using) a wild-type immunoglobulin hingeregion that contains one or more cysteine residues. In certainembodiments, a mutated or otherwise altered hinge region polypeptide maycontain zero or only one cysteine residue, wherein the mutated orotherwise altered hinge region polypeptide is or has been derived from awild type immunoglobulin hinge region that contains, respectively, oneor more or two or more cysteine residues. In the mutated or otherwisealtered hinge region polypeptide, the cysteine residues of the wild-typeimmunoglobulin hinge region are preferably deleted or substituted withamino acids that are incapable of forming a disulfide bond. In oneembodiment of the invention, a mutated or otherwise altered hinge regionpolypeptide is or has been derived from a human IgG wild-type hingeregion polypeptide, which may include any of the four human IgG isotypesubclasses, IgG1, IgG2, IgG3 or IgG4. In certain preferred embodiments,the mutated or otherwise altered hinge region polypeptide is or has beenderived from (or using) a human IgA or IgD wild-type hinge regionpolypeptide. By way of example, a mutated or otherwise altered hingeregion polypeptide that is or has been derived from a human IgG1 or IgAwild-type hinge region polypeptide may comprise mutations, alterations,or deletions at two of the three cysteine residues in the wild-typeimmunoglobulin hinge region, or mutations, alterations, or deletions atall three cysteine residues.

The cysteine residues that are present in a wild-type immunoglobulinhinge region and that are removed or altered by mutagenesis or any othertechniques according to particularly preferred embodiments of thepresent invention include cysteine residues that form, or that arecapable of forming, interchain disulfide bonds. Without wishing to bebound by particular theory or mechanism of action, the present inventioncontemplates that mutation, deletion, or other alteration of such hingeregion cysteine residues, which are believed to be involved in formationof interchain disulfide bridges, reduces the ability of the subjectinvention binding domain-immunoglobulin fusion protein to dimerize (orform higher oligomers) via interchain disulfide bond formation, whilesurprisingly not ablating or undesireably compromising the ability of afusion protein or other construct to promote ADCC, and/or CDC and/or tofix complement. In particular, the Fc receptors that mediate ADCC (e.g.,FcRIII, CD16) exhibit low affinity for immunoglobulin Fc domains,supporting the idea that functional binding of Fc to FcR requiresavidity stabilization of the Fc-FcR complex by virtue of the dimericstructure of heavy chains in a conventional antibody, and/or FcRaggregation and cross-linking by a conventional antibody Fc structure.Sonderman et al., 2000 Nature 406:267; Radaev et al., 2001 J. Biol.Chem. 276:16469; Radaev et al., 2001 J. Biol. Chem. 276:16478; Koolwijket al., 1989 J. Immunol. 143:1656; Kato et al., 2000 Immunol. Today21:310. Hence, the constructs, including for example bindingdomain-immunoglobulin fusion proteins, of the present invention providethe advantages associated with single-chain constructs includingsinge-chain immunoglobulin fusion proteins while also unexpectedlyretaining one or more immunological activities. Similarly, the abilityto fix complement is typically associated with immunoglobulins that aredimeric with respect to heavy chain constant regions such as those thatcomprise Fc, while various constructs, including bindingdomain-immunoglobulin fusion proteins, of the present invention, whichmay, due to the replacement or deletion of hinge region cysteineresidues or due to other structural modifications as described herein,for example, have compromised or ablated abilities to form interchaindisulfide bonds, exhibit the unexpected ability to fix complement.Additionally, according to certain embodiments of the present inventionwherein a construct, including, for example, a bindingdomain-immunoglobulin fusion protein, may comprise a connecting regionand tail region comprising, or consisting essentially of, or consistingof, one or more of a human IgE hinge-acting region, i.e., a IgE CH2region polypeptide, a human IgE CH3 constant region polypeptide, and ahuman IgE CH4 constant region polypeptide, the invention constructsincluding fusion proteins unexpectedly retain the immunological activityof mediating ADCC and/or of inducing an allergic response mechanism.

Selection of an immunoglobulin hinge region polypeptide as a connectingregion according to certain embodiments of the subject inventionconstructs, such as binding domain-immunoglobulin fusion proteins, mayrelate to the use of an “alternative hinge region” polypeptide sequence,which includes a polypeptide sequence that is not necessarily derivedfrom any immunoglobulin hinge region sequence per se. Instead, analternative hinge region refers to a hinge region polypeptide thatcomprises an amino acid sequence, or other molecular sequence, of atleast about ten consecutive amino acids or molecules, and in certainembodiments at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-25, 26-30, 31-50, 51-60, 71-80, 81-90, or 91-110 amino acids ormolecules that is present in a sequence disclosed herein, for example apolypeptide sequence that is or has been derived from a region locatedbetween intrachain disulfide-generated immunoglobulin-like loop domainsof immunoglobulin gene superfamily members such as CD2 (e.g., GenbankAcc. No. NM_(—)001767), CD4 (e.g., Genbank Acc. No. NM_(—)000616), CD5(e.g., Genbank Acc. No. BCO27901), CD6 (e.g., Genbank Acc. No.NM_(—)006725), CD7 (e.g., Genbank Acc. Nos. XM_(—)046782, BC009293,NM_(—)006137) or CD8 (e.g., Genbank Acc. No. M12828), or other Igsuperfamily members. By way of non-limiting example, an alternativehinge region used as a connecting region, for example, may provide aglycosylation site as provided herein, or may provide a humangene-derived polypeptide sequence for purposes of enhancing the degreeof “humanization” of a fusion protein, or may comprise, or consistessentially of, or consist of, an amino acid sequence that eliminates orreduces the ability of a construct of the invention, such as a fusionprotein, to form multimers or oligomers or aggregates or the like.Certain alternative hinge region polypeptide sequences, including thosedescribed herein, may be derived or constructed from (or using) thepolypeptide sequences of immunoglobulin gene superfamily members thatare not actual immunoglobulins per se. For instance and according tonon-limiting theory, certain polypeptide sequences that are situatedbetween intrachain disulfide-generated immunoglobulin loop domain ofimmunoglobulin gene super-family member proteins may be used in whole orin part as alternative hinge region polypeptides as provided herein, ormay be further modified for such use.

As noted above, the constructs of the invention, including bindingdomain-immunoglobulin fusion proteins, are believed, according tonon-limiting theory, to be compromised in their ability to dimerize viainterchain disulfide bond formation, and further according to theory,this property is a consequence, in whole or in part, of a reduction inthe number of cysteine residues that are present in an immunoglobulinhinge region polypeptide selected for inclusion in the construction ofthe construct, such as a fusion protein construct. Determination of therelative ability of a polypeptide to dimerize is well within theknowledge of the relevant art, where any of a number of establishedmethodologies may be applied to detect protein dimerization (see, e.g.,Scopes, Protein Purification: Principles and Practice, 1987Springer-Verlag, New York). For example, biochemical separationtechniques for resolving proteins on the basis of molecular size (e.g.,gel electrophoresis, gel filtration chromatography, analyticalultracentrifugation, etc.), and/or comparison of protein physicochemicalproperties before and after introduction of sulfhydryl-active (e.g.,iodoacetamide, N-ethylmaleimide) or disulfide-reducing (e.g.,2-mercaptoethanol, dithiothreitol) agents, or other equivalentmethodologies, may all be employed for determining a degree ofpolypeptide dimerization or oligomerization, and for determiningpossible contribution of disulfide bonds to such potential quarternarystructure. In certain embodiments, the invention relates to a construct,for example a binding domain-immunoglobulin fusion protein, thatexhibits a reduced (i.e., in a statistically significant manner relativeto an appropriate IgG-derived control, for example) ability to dimerize,relative to a wild-type human immunoglobulin G hinge region polypeptideas provided herein. Those familiar with the art will be able readily todetermine whether a particular fusion protein displays such reducedability to dimerize.

Compositions and methods for preparation of immunoglobulin fusionproteins, for example, are well known in the art. See, e.g., U.S. Pat.No. 5,892,019, which reports recombinant proteins that are the productof a single encoding polynucleotide but which are not constructs,including binding domain-immunoglobulin fusion proteins, according tothe present invention.

For a construct, for example, in an immunoglobulin fusion protein of theinvention that is intended for use in humans, any included Ig constantregions will typically be of human sequence origin, or humanized, tominimize a potential anti-human immune response and to provideappropriate and/or desired effector functions. Manipulation of sequencesencoding antibody constant regions is referenced in the PCT publicationof Morrison and Oi, WO 89/07142. In particularly preferred embodiments,a tail region is prepared from an immunoglobulin heavy chain constantregion in which the CH1 domain is or has been deleted (the CH1 and CH2regions in the case of IgE) and the carboxyl end of the binding domain,or where the binding domain comprises two immunoglobulin variable regionpolypeptides, the second (i.e., more proximal to the C-terminus)variable region is joined to the amino terminus of CH2 through one ormore connecting regions, such as a hinge or altered region. A schematicdiagram depicting the structures of two exemplary bindingdomain-immunoglobulin fusion proteins is shown in FIG. 11. Inparticularly preferred embodiments no interchain disulfide bonds arepresent, and in other embodiments a restricted number of interchaindisulfide bonds may be present relative to the number of such bonds thatwould be present if wild-type hinge region polypeptides were insteadpresent. In other embodiments a construct of the invention, such as forexample, a fusion protein, comprises, or consists essentially of, orconsists of, a mutated or otherwise altered hinge region polypeptidethat exhibits a reduced ability to dimerize, relative to a wild-typehuman IgG hinge region polypeptide. Thus, an isolated polynucleotidemolecule coding for such a single chain construct, such as animmunoglobulin fusion protein, has a binding region, for example, adomain that provides specific or otherwise desired binding affinity andselectivity for a target, such as a target antigen.

The invention also contemplates, for example, in certain embodiments,constructs including binding domain-immunoglobulin fusion proteins thatcomprise fused or otherwise connected polypeptide sequences or portionsthereof derived or prepared from a plurality of genetic sources, forexample, according to molecular “domain swapping” paradigms. Thosehaving familiarity with the art will appreciate that selection of suchpolypeptide sequences for assembly into a construct, such as a bindingdomain-immunoglobulin fusion protein, for example, may involvedetermination of appropriate portions of each component polypeptidesequence, for example, based on structural and/or functional propertiesof each such sequence (see, e.g., Carayannopoulos et al., 1996 J. Exp.Med. 183:1579; Harlow et al., Eds., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor (1988)). The componentpolypeptide sequences of which the construct, such as a fusion protein,is comprised or prepared may therefore comprise intact or full-lengthbinding domain, immunoglobulin, linker and/or plasma membrane anchordomain polypeptide sequences, or truncated versions or variants thereofsuch as those provided herein. According to these and relatedembodiments of the invention, any two or more of the candidate componentpolypeptides of which the subject invention constructs, for example,fusion proteins, may be comprised will be derived or prepared fromindependent sources, such as from immunoglobulin sequences of differingallotype, isotype, subclass, class, or species of origin (e.g.,xenotype). Thus, as a non-limiting example, a binding domain polypeptide(or its constituent polypeptides such as one or more variable regionpolypeptides and/or a linker polypeptide), a hinge region polypeptide,immunoglobulin heavy chain CH2 and CH3 constant region polypeptides andoptionally an immunoglobulin heavy chain CH4 constant region polypeptideas may be obtained from an IgM or IgE heavy chain, and a plasma membraneanchor domain polypeptide may all be separately obtained from distinctgenetic sources and engineered into a chimeric or fusion protein usingwell known techniques and according to methodologies described herein,for example.

Accordingly, a construct of the invention, for example a bindingdomain-immunoglobulin fusion protein according to certain embodiments ofthe present invention, may also therefore comprise in pertinent part animmunoglobulin heavy chain CH3 constant region polypeptide that is awild-type IgA CH3 constant region polypeptide, or alternatively, that isa mutated or otherwise altered or substituted or truncated IgA CH3constant region polypeptide that is incapable of associating with a Jchain, or that will not associate to an undesired degree with a J chain;preferably the IgA CH3 constant region polypeptides used in a tailregion portion of a construct are of human origin or are humanized. Byway of brief background, IgA molecules are known to be released intosecretory fluids by a mechanism that involves association of IgA intodisulfide-linked polymers (e.g., dimers) via a J chain polypeptide(e.g., Genbank Acc. Nos. XM_(—)059628, M12378, M12759; Johansen et al.,1999 Eur. J. Immunol. 29:1701) and interaction of the complex so formedwith another protein that acts as a receptor for polymericimmunoglobulin, and which is known as transmembrane secretory component(SC; Johansen et al., 2000 Sc. J. Immunol. 52:240; see also, e.g.,Sorensen et al., 2000 Int. Immunol. 12:19; Yoo et al., 1999 J. Biol.Chem. 274:33771; Yoo et al., 2002 J. Immunol. Meth. 261:1; Corthesy,2002 Trends Biotechnol. 20:65; Symersky et al., 2000 Mol. Immunol.37:133; Crottet et al., 1999 Biochem. J. 341:299). Interchain disulfidebond formation between IgA Fc domains and J chain is mediated through apenultimate cysteine residue in an 18-amino acid C-terminal extensionthat forms part of the IgA heavy chain constant region CH3 domainpolypeptide (Yoo et al., 1999; Sorensen et al., 2000). Certainembodiments of the subject invention constructs, including for example,fusion proteins, therefore contemplate inclusion of the wild-type IgAheavy chain constant region polypeptide sequence, which is capable ofassociating with J chain. Certain other embodiments of the invention,however, contemplate fusion proteins that comprise a mutated orotherwise altered, substituted, or truncated IgA CH3 constant regionpolypeptide that is incapable of associating with a J chain. Accordingto such embodiments, for example, two or more residues from theC-terminus of an IgA CH3 constant region polypeptide such as a human IgACH3 constant region polypeptide may be deleted to yield a truncated CH3constant region polypeptide as provided herein. In preferred embodimentsand as described in greater detail herein, a mutated human IgA CH3constant region polypeptide that is incapable of associating with a Jchain comprises such a C-terminal deletion of either four or 18 aminoacids. However, the invention need not be so limited, such that themutated IgA CH3 constant region polypeptide may comprise a deletion ofabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21-25, 26-30 or more amino acids, so long as the construct, forexample, the fusion protein, is capable of specifically binding anantigen and of at least one immunological activity as provided herein.Alternatively, the invention also contemplates constructs, for example,fusion proteins, having a tail region that comprises a mutated IgA CH3constant region polypeptide that is incapable of associating with a Jchain by virtue of replacement of the penultimate cysteine, or bychemical modification of that amino acid residue, in a manner thatprevents, or inhibits an undesired level of, interchain disulfide bondor multimer formation. Methods for determining whether a construct, forexample a fusion protein, can associate with a J chain will be known tothose having familiarity with the art and are described or referencedherein.

As also described herein and according to procedures known in the art,the construct, for example a fusion protein, may further be testedroutinely for immunological activity, for instance, in ADCC or CDCassays. As an illustrative example, a construct, for example a fusionprotein, according to such an embodiment may comprise a binding domainpolypeptide derived or constructed from (or using) a native orengineered human heavy chain variable region polypeptide sequence, anative or engineered human IgA-derived immunoglobulin hinge regionpolypeptide sequence, a native or engineered human IgG1 immunoglobulinheavy chain CH2 constant region polypeptide sequence, a native orengineered human IgG2 immunoglobulin heavy chain CH3 constant regionpolypeptide sequence, and optionally a native or engineered human IgEimmunoglobulin heavy chain CH4 constant region polypeptide sequenceand/or a native or engineered human TNF-α receptor type 1 (TNFR1) plasmamembrane anchor domain polypeptide sequence that comprises a cytoplasmictail polypeptide which is capable of apoptotic signaling or otherwisepromoting apoptosis. The invention therefore contemplates these andother embodiments according to the present invention in which two ormore polypeptide sequences that are present in a construct, for examplea fusion protein, have independent genetic origins.

As noted above, in certain embodiments the construct, for example abinding protein-immunoglobulin fusion protein, comprises at least onenative or engineered immunoglobulin variable region polypeptide, whichmay be a native or engineered light chain or a native or engineeredheavy chain variable region polypeptide, and in certain embodiments thefusion protein comprises at least one such native or engineered lightchain V-region and one such native or engineered heavy chain V-regionand at least one linker peptide that is fused or otherwise connected toto each of the native or engineered V-regions. Construction of suchbinding domains, for example single chain Fv domains, is known in theart and is described in greater detail in the Examples below, and hasbeen described, for example, in various documents cited herein;selection and assembly of single-chain variable regions and of linkerpolypeptides that may be fused or otherwise connected to each of a heavychain-derived and a light chain-derived V region (e.g., to generate abinding region, such as a binding domain that comprises a single-chainFv polypeptide) is also known to the art and described herein. See,e.g., U.S. Pat. Nos. 5,869,620, 4,704,692, and 4,946,778. In certainembodiments all or a portion or portions of an immunoglobulin sequencethat is derived from a non-human source may be “humanized” according torecognized procedures for generating humanized antibodies, i.e.,immunoglobulin sequences into which human Ig sequences are introduced toreduce the degree to which a human immune system would perceive suchproteins as foreign (see, e.g., U.S. Pat. Nos. 5,693,762; 5,585,089;4,816,567; 5,225,539; 5,530,101; and documents cited therein).

Constructs of the invention, including binding domain-immunoglobulinfusion proteins, as described herein may, according to certainembodiments, desirably comprise sites for glycosylation, e.g., covalentattachment of carbohydrate moieties such as, for example,monosaccharides or oligosaccharides. Incorporation of amino acidsequences that provide substrates for polypeptide glycosylation iswithin the scope of the relevant art, including, for example, the use ofgenetic engineering or protein engineering methodologies to obtain apolypeptide sequence containing, for example, the classic Asn-X-Ser/Thrsite for N-(asparagine)-linked glycosylation, or a sequence containingSer or Thr residues that are suitable substrates for O-linkedglycosylation, or sequences amenable to C-mannosylation,glypiation/glycosylphosphatidylinositol modification, orphosphoglycation, all of which can be identified according toart-established criteria (e.g., Spiro, 2002 Glybiology 12:43R). Withoutwishing to be bound by any particular theory or mechanism, glycosylatedconstructs such as fusion proteins having particular amino acidsequences may beneficially possess attributes associated with one ormore of improved solubility, enhanced stability in solution, enhancedphysiological stability, improved bioavailability including in vivobiodistribution, and superior resistance to proteases, all in astatistically significant manner, relative to constructs, includingfusion proteins, having the same or highly similar amino acid sequencesbut lacking glycosyl moieties. In certain preferred embodiments thesubject invention constructs, such as fusion protein constructs, maycomprise a glycosylation site that is present in a linker as providedherein, and in certain other preferred embodiments the subject inventionconstruct, for example, a fusion protein, comprises a glycosylation sitethat is present in a connecting region, such as a hinge regionpolypeptide sequence as provided herein.

In certain preferred embodiments of the present invention, such as thoseuseful for gene therapy applications or in display systems or assays,such as screening assays (including library display systems and libraryscreening assays), the construct, for example, a bindingdomain-immunoglobulin fusion protein, is a protein or glycoprotein thatis capable of being expressed by a host cell such that it localizes tothe cell surface. Constructs, such as binding domain-immunoglobulinfusion proteins, that localize to the cell surface may do so by virtueof having naturally present or artificially introduced structuralfeatures that direct the fusion protein to the cell surface (e.g.,Nelson et al. 2001 Trends Cell Biol. 11:483; Ammon et al., 2002 Arch.Physiol. Biochem. 110:137; Kasai et al., 2001 J. Cell Sci. 114:3115;Watson et al., 2001 Am. J. Physiol. Cell Physiol. 281:C215; Chatterjeeet al., 200 J. Biol. Chem. 275:24013) including by way of illustrationand not limitation, secretory signal sequences, leader sequences, plasmamembrane anchor domain polypeptides and transmembrane domains such ashydrophobic transmembrane domains (e.g., Heuck et al., 2002 CellBiochem. Biophys. 36:89; Sadlish et al., 2002 Biochem J. 364:777;Phoenix et al., 2002 Mol. Membr. Biol. 19:1; Minke et al., 2002 Physiol.Rev. 82:429) or glycosylphosphatidylinositol attachment sites(“glypiation” sites, e.g., Chatterjee et al., 2001 Cell Mol. Life. Sci.58:1969; Hooper, 2001 Proteomics 1:748; Spiro, 2002 Glycobiol. 12:43R),cell surface receptor binding domains, extracellular matrix bindingdomains, or any other structural feature that causes at least a desiredportion of the fusion protein population to localize, in whole or inpart, to the cell surface. Particularly preferred are fusion proteinconstructs that comprise a plasma membrane anchor domain which includesa transmembrane polypeptide domain, typically comprising a membranespanning domain which includes a hydrophobic region capable ofenergetically favorable interaction with the phospholipid fatty acyltails that form the interior of the plasma membrane bilayer. Suchfeatures are known to those of ordinary skill in the art, who willfurther be familiar with methods for introducing nucleic acid sequencesencoding these features into the subject expression constructs bygenetic engineering, and with routine testing of such constructs toverify cell surface localization of the product.

According to certain further embodiments, a plasma membrane anchordomain polypeptide comprises such a transmembrane domain polypeptide andalso comprises a cytoplasmic tail polypeptide, which refers to a regionor portion of the polypeptide sequence that contacts the cytoplasmicface of the plasma membrane and/or is in contact with the cytosol orother cytoplasmic components. A large number of cytoplasmic tailpolypeptides are known that comprise the intracellular portions ofplasma membrane transmembrane proteins, and discrete functions have beenidentified for many such polypeptides, including biological signaltransduction (e.g., activation or inhibition of protein kinases, proteinphosphatases, G-proteins, cyclic nucleotides and other secondmessengers, ion channels, secretory pathways), biologically activemediator release, stable or dynamic association with one or morecytoskeletal components, cellular differentiation, cellular activation,mitogenesis, cytostasis, apoptosis and the like (e.g., Maher et al.,2002 Immunol. Cell Biol. 80:131; El Far et al., 2002 Biochem J. 365:329;Teng et al., 2002 Genome Biol. 2REVIEWS:3012; Simons et al., 2001 CellSignal 13:855; Furie et al., 2001 Thromb. Haemost. 86:214; Gaffen, 2001Cytokine 14:63; Dittel, 2000 Arch. Immunol. Ther. Exp. (Warsz.) 48:381;Parnes et al., 2000 Immunol. Rev. 176:75; Moretta et al., 2000 Semin.Immunol. 12:129; Ben Ze'ev, 1999 Ann. N.Y. Acad. Sci. 886:37; Marsterset al., Recent Prog. Horm. Res. 54:225).

FIG. 70 illustrates the binding, for example, of fluorceine-conjugatedFcRIII (CD16) soluble fusion proteins to 2H7 scFv-binding domainconstructs that are attached to CD20 expressed by cells, CHO cells inthis example. CD16 binding to a construct of the invention, for example,scFv-binding domain construct, provides one example of a screening toolthat may be used to detect and/or quantitate changes in CD16 binding toaltered constructs of the invention, including scFv-binding domainconstructs, that contains targeted or site-specific mutations,substitutions, deletions, or other alterations. Changes in CD16 bindingproperties may be reflected, for example, by changes in binding ofeither CD16 high affinity protein (158V) or CD16 low affinity protein(158 F) or both.

A schematic representation of one example of such a screening process isdiagrammed in the second drawing in FIG. 70, in which scFv-bindingdomain constructs are displayed on the cell surface of mammalian cells.scFv-binding domain molecules in this example are displayed on the cellsurface through a molecule that serves as a transmembrane domain anchor.These molecules may represent, for example, a single scFv-binding domainconstruct or may be introduced into a population of mammalian cells as alibrary of such molecules. Transfected cells with altered bindingproperties can then, for example, be panned, sorted, or otherwiseisolated from other cells by altering the stringency of the selectionconditions and using CD16 fusion proteins as a binding probe. Cells thatexpress scFv-Ig molecules with altered binding to either CD16 highaffinity allele (158V) or CD16 low affinity allele (158F) or both, forexample, can be isolated.

This display system can be used, for example, to create a library ofconstructs of the invention with mutated or otherwise altered tailregions with short stretches of CH2 sequence replaced with randomizedoligonucleotides or, for example, randomization of a single residue withall possible amino acid substitutions, natural or unnatural, includingsynthetic amino acids. Once such a library is constructed, it can betransfected into appropriate cells, for example, COS cells, by methodsknown in the art. Transfectants can then be bound to, for example,labeled CD16 constructs, and panned or sorted based on their relative ordesired binding properties to multiple allelotypes/isoforms. Desiredcells may be harvested, and the DNA, for example, plasmid DNA, isolatedand then transformed into, for example, bacteria. This process may berepeated iteratively multiple times until desire single clones areisolated from the mammalian host cells. See Seed B and Aruffo A, Pro.Nat'l Acad Sci USA 1987 84:3365-3369; Aruffo A and Seed B, Pro. Nat'lAcad Sci USA 1987 84:8573-8577.

One such use of this type of screening system, for example, is for theidentification and/or isolation of constructs of the invention havingtail regions, or tail regions, that bind equally well to both the highand low affinity alleles of CD16 with the goal of improving effectorfunctions mediated by scFv-binding domain constructs in multiplesubpopulations of patients. Constructs of the invention having tailregions, or tail regions with altered binding properties to other Fcreceptors can also be selected using such a display system, for example,the display system described. Other display systems that do notglycosylate proteins, for example, those that use bacteriophage oryeast, are not generally desired for selection of constructs of theinvention having Ig-based tail regions, or Ig-based tail regions, withaltered FcR binding properties. Most non-mammalian systems, for example,do not glycosylate proteins.

Expression of constructs of the invention, for example, scFv-bindingdomain constructs, expressd on the surface of a mammalian cell byincorporation of an appropriate molecule into the construct, forexample, by incorporation of a transmembrane domain or a GPI anchorsignal, also have utility in other display systems that are useful, forexample, for selection of constructs of the invention, for example,altered scFv-binding domain molecules that will be produced at higher orother desired levels. In one such an embodiment, cells that are usefulin the production of glycosylated proteins, for example, mammalian cellssuch as COS cells, are transfected with a library of scFv-binding domainconstructs in a plasmid that directs their expression to the cellsurface. Cells, such as COS cells, that express the highest or otherdesired level of the scFv-binding domain molecules are selected bytechniques known in the art (for example panning, sterile cell sorting,magnetic bead separation, etc.), and DNA, for example, plasmid DNA, isisolated for transformation into other cells, for example, bacteria.After one or more rounds of selection single clones are isolated thatencode scFv-binding domain molecules capable of a high or other desiredlevel of expression. The isolated clones may then be altered to removethe membrane anchor and expressed in an appropriate cells system, forexample, a mammalian cell system, wherein the scFv-binding domainconstructs will be produced, for example, by secretion into the culturefluid at desired levels. Without being bound by any particular mechanismor theory, this is believed to result from the common requirement ofsecreted glycoproteins and cell surface glycoproteins for a signalpeptide and processing through the golgi for expression. Thus, selectionfor a molecule that shows an improvement expression levels on a cellsurface will also result in the identification of a molecule having animprovement in levels of secreted protein.

These display systems utilizing a construct of the invention may also beused for screening and/or identifying and/or isolating affinity variantsof the binding domain within a construct.

Particularly preferred are such display and/or screening systems, forexample, that include or use constructs that include (1) animmunoglobulin variable region polypeptide sequence, including native orengineered V_(H) and/or V_(L) and/or single-chain variable region (sFv)sequences, and which include, for example, a mutation, alteration ordeletion at an amino acid at a location or locations corresponding toone or more of amino acid positions 9, 10, 11, 12, 108, 110, 111, and112, in a V_(H) region sequence (including in a V_(H) region sequencewithin an scFv or other construct), and/or (2) an immunoglobulinvariable region polypeptide sequence, including native or engineeredV_(H) and/or V_(L) and/or single-chain variable region (sFv) sequences,and which include, for example, a mutation, alteration or deletion at alocation or locations corresponding to one or more of amino acidpositions 12, 80, 81, 82, 83, 105, 106, 107 and 108 in a light chainvariable region sequence (including in a V_(L) region sequence within anscFv or other construct). Especially preferred are such display and/orscreening systems that include or use constructs that include anengineered V_(H) sequence (whether or not associated with one or moreother sequences, including immunoglobulin-derived and other sequencescontained, for example, within an sFv or scFv-containing construct),which includes a mutation, alteration or deletion at an amino acid at alocation or locations corresponding to amino acid position 11. TheV_(H)11 amino acid, if substituted, may be substituted with anotheramino acid as described herein, or by another molecule as desired.

In the context of other methods of using constructs of the invention,including binding domain-immunoglobulin fusion proteins, for thetreatment of a malignant condition or a B cell disorder(s) as providedherein, including, for example, by one or more of a number of genetherapy methods and related construct delivery techniques, the presentinvention also contemplates certain embodiments wherein a construct, forexample, a binding domain-immunoglobulin fusion protein that comprises aplasma membrane anchor domain polypeptide is expressed (or capable orexpression) at a cell surface and may further comprise a cytoplasmictail polypeptide which comprises an apoptosis signaling polypeptidesequence. A number of apoptosis signaling polypeptide sequences areknown to the art, as reviewed, for example, in When Cells Die: AComprehensive Evaluation of Apoptosis and Programmed Cell Death (R. A.Lockshin et al., Eds., 1998 John Wiley & Sons, New York; see also, e.g.,Green et al., 1998 Science 281:1309 and references cited therein;Ferreira et al., 2002 Clin. Canc. Res. 8:2024; Gurumurthy et al., 2001Cancer Metastas. Rev. 20:225; Kanduc et al., 2002 Int. J. Oncol.21:165). Typically an apoptosis signaling polypeptide sequence comprisesall or a portion of, or is derived or constructed from, a receptor deathdomain polypeptide, for instance, FADD (e.g., Genbank Acc. Nos. U24231,U43184, AF009616, AF009617, NM_(—)012115), TRADD (e.g., Genbank Acc. No.NM_(—)003789), RAIDD (e.g., Genbank Acc. No. U87229), CD95 (FAS/Apo-1;e.g., Genbank Acc. Nos. X89101, NM_(—)003824, AF344850, AF344856),TNF-α-receptor-1 (TNFR1, e.g., Genbank Acc. Nos. 563368, AF040257), DR5(e.g., Genbank Acc. No. AF020501, AF016268, AF012535), an ITIM domain(e.g., Genbank Acc. Nos. AF081675, BC015731, NM_(—)006840, NM_(—)006844,NM_(—)006847, XM_(—)017977; see, e.g., Billadeau et al., 2002 J. Clin.Invest. 109:161), an ITAM domain (e.g., Genbank Acc. Nos. NM_(—)005843,NM_(—)003473, BC030586; see, e.g., Billadeau et al., 2002), or otherapoptosis-associated receptor death domain polypeptides known to theart, for example, TNFR2 (e.g., Genbank Acc. No. L49431, L49432),caspase/procaspase-3 (e.g., Genbank Acc. No. XM_(—)54686),caspase/procaspase-8 (e.g., AF380342, NM_(—)004208, NM_(—)001228,NM_(—)033355, NM_(—)033356, NM_(—)033357, NM_(—)033358),caspase/procaspase-2 (e.g., Genbank Acc. No. AF314174, AF314175), etc.

Cells in biological samples that are suspected of undergoing apoptosismay be examined for morphological, permeability or other changes thatare indicative of an apoptotic state. For example by way of illustrationand not limitation, apoptosis in many cell types may cause alteredmorphological appearance such as plasma membrane blebbing, cell shapechange, loss of substrate adhesion properties or other morphologicalchanges that can be readily detected by a person having ordinary skillin the art, for example by using light microscopy. As another example,cells undergoing apoptosis may exhibit fragmentation and disintegrationof chromosomes, which may be apparent by microscopy and/or through theuse of DNA-specific or chromatin-specific dyes that are known in theart, including fluorescent dyes. Such cells may also exhibit alteredplasma membrane permeability properties as may be readily detectedthrough the use of vital dyes (e.g., propidium iodide, trypan blue) orby the detection of lactate dehydrogenase leakage into the extracellularmilieu. These and other means for detecting apoptotic cells bymorphologic criteria, altered plasma membrane permeability, and relatedchanges will be apparent to those familiar with the art.

In another embodiment of the invention wherein a construct, such as abinding domain-immunoglobulin fusion protein, that is expressed at acell surface comprises a plasma membrane anchor domain having atransmembrane domain and a cytoplasmic tail that comprises an apoptosissignaling polypeptide, cells in a biological sample may be assayed fortranslocation of cell membrane phosphatidylserine (PS) from the inner tothe outer leaflet of the plasma membrane, which may be detected, forexample, by measuring outer leaflet binding by the PS-specific proteinannexin. Martin et al., J. Exp. Med. 182:1545, 1995; Fadok et al., J.Immunol. 148:2207, 1992. In still other related embodiments of theinvention, including embodiments wherein a construct, such as a bindingdomain-immunoglobulin fusion protein, is expressed at the cell surfaceand comprises a plasma membrane anchor domain having an apoptosissignaling polypeptide and also including embodiments wherein theconstruct, such as a binding domain-immunoglobulin fusion protein, is asoluble protein that lacks a membrane anchor domain and that is capableof inducing apoptosis, a cellular response to an apoptogen is determinedby an assay for induction of specific protease activity in any member ofa family of apoptosis-activated proteases known as the caspases (see,e.g., Green et al., 1998 Science 281:1309). Those having ordinary skillin the art will be readily familiar with methods for determining caspaseactivity, for example by determination of caspase-mediated cleavage ofspecifically recognized protein substrates. These substrates mayinclude, for example, poly-(ADP-ribose) polymerase (PARP) or othernaturally occurring or synthetic peptides and proteins cleaved bycaspases that are known in the art (see, e.g., Ellerby et al., 1997 J.Neurosci. 17:6165). The synthetic peptide Z-Tyr-Val-Ala-Asp-AFC (SEQ IDNO:528;), wherein “Z” indicates a benzoyl carbonyl moiety and AFCindicates 7-amino-4-trifluoromethylcoumarin (Kluck et al., 1997 Science275:1132; Nicholson et al., 1995 Nature 376:37), is one such substrate.Other non-limiting examples of substrates include nuclear proteins suchas U1-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, 1997 J. Cell.Biochem. 64:50; Cohen, 1997 Biochem. J. 326:1). Cellular apoptosis mayalso be detected by determination of cytochrome c that has escaped frommitochondria in apoptotic cells (e.g., Liu et al., Cell 86:147, 1996).Such detection of cytochrome c may be performed spectrophotometrically,immunochemically or by other well established methods for determiningthe presence of a specific protein. Persons having ordinary skill in theart will readily appreciate that there may be other suitable techniquesfor quantifying apoptosis.

Particularly preferred embodiments of constructs useful for gene therapyapplications, including those constructs that include a plasma membraneanchor domain and/or cytoplasmic tail polypeptide (including, forexample, an apoptosis signaling sequence), are such constructs thatinclude (1) an immunoglobulin variable region polypeptide sequence,including native or engineered V_(H) and/or V_(L) and/or single-chainvariable region (sFv) sequences, and which include, for example, amutation, alteration or deletion at an amino acid at a location orlocations corresponding to one or more of amino acid positions 9, 10,11, 12, 108, 110, 111, and 112, in a V_(H) region sequence (including ina V_(H) region sequence within an scFv or other construct), and/or (2)an immunoglobulin variable region polypeptide sequence, including nativeor engineered V_(H) and/or V_(L) and/or single-chain variable region(sFv) sequences, and which include, for example, a mutation, alterationor deletion at a location or locations corresponding to one or more ofamino acid positions 12, 80, 81, 82, 83, 105, 106, 107 and 108 in alight chain variable region sequence (including in a V_(L) regionsequence within an scFv or other construct). Especially preferred areconstructs that include an engineered V_(H) sequence (whether or notassociated with one or more other sequences, includingimmunoglobulin-derived and other sequences contained, for example,within an sFv or scFv-containing construct), which includes a mutation,alteration or deletion at an amino acid at a location or locationscorresponding to amino acid position 11. The V_(H)11 amino acid, ifsubstituted, may be substituted with another amino acid as describedherein, or by another molecule as desired.

Once a construct, such as for example a binding domain-immunoglobulinfusion protein, as provided herein has been designed, polynucleotidesincluding DNAs encoding the construct, where it or a relevant portion ofit is a polypeptide, may be synthesized in whole or in part viaoligonucleotide synthesis as described, for example, in Sinha et al.,Nucleic Acids Res., 12:4539-4557 (1984); assembled via PCR as described,for example in Innis, Ed., PCR Protocols, Academic Press (1990) and alsoin Better et al. J. Biol. Chem. 267:16712-16118 (1992); cloned andexpressed via standard procedures as described, for example, in Ausubelet al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons,New York (1989) and also in Robinson et al., Hum. Antibod. Hybridomas,2:84-93 (1991); and tested for desired activity, for example, binding toa target, or specific antigen binding activity, as described, forexample, in Harlow et al., Eds., Antibodies: A Laboratory Manual,Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988) andMunson et al., Anal. Biochem., 107:220-239 (1980).

The preparation of single polypeptide chain binding molecules of the Fvregion, single-chain Fv molecules, is known in the art. See, e.g., U.S.Pat. No. 4,946,778. In the present invention, single-chain Fv-likemolecules that may be included in constructs of the invention may besynthesized by encoding a first variable region of the heavy or lightchain, followed by one or more linkers to the variable region of thecorresponding light or heavy chain, respectively. The selection ofvarious appropriate linker(s) between the two variable regions isdescribed in U.S. Pat. No. 4,946,778 (see also, e.g., Huston et al.,1993 Int. Rev. Immunol. 10:195). An exemplary linker described herein is(Gly-Gly-Gly-Gly-Ser)₃ (SEQ ID NO:529), but may be of any desiredlength. The linker is used to convert the naturally aggregated butchemically separate heavy and light chains into the amino terminalantigen binding portion of a single polypeptide chain, for example,wherein this antigen binding portion will fold into a structure similarto the original structure made of two polypeptide chains, or thatotherwise has the ability to bind to a target, for example a targetantigen. For those constructs that include an scFv as a binding region,a native or engineered immunoglobulin hinge as a connecting region, andone or more native or engineered heavy chain constant regions as abinding region, nucleotide sequences encoding the variable regions ofnative or engineered heavy and light chains, joined by a sequenceencoding a linker, are joined to a nucleotide sequence encoding nativeor engineeredantibody constant regions, as desired. The constant regionsmay be those that permit the resulting polypeptide to form interchaindisulfide bonds to form a dimer, and which contain desired effectorfunctions, such as the ability to mediate ADCC, CDC, or fix complement,although native or engineered constant regions that do not favor dimeror other multimer fomation or aggregation are preferred. For aconstruct, such as an immunoglobulin-like molecule, of the inventionthat is intended for use in humans, the included sequences havingconstant regions and/or desired constant regions function(s) willtypically be human or substantially human or humanized to minimize apotential anti-human immune response and to provide appropriate ordesired effector functions. Manipulation of sequences encoding antibodyconstant regions is referenced in the PCT publication of Morrison andOi, WO 89/07142. In preferred embodiments, the CH1 domain is deleted inwhole or in part from a tail region that includes, or consistsessentially of, or consists of, a native or engineered immunoglobulinconstant region(s) (for example, native or engineered CH2 and/or CH3constant region(s), or native or engineered CH2 and/or CH3 and/or CH4constant region(s)), and the carboxyl end of the binding region, forexample, a binding domain polypeptide such as an immunoglobulin variableregion polypeptide, is joined to the amino terminus of, for example, aCH2 via a connecting region, for example, a native or engineered hingeregion polypeptide as provided herein.

As described above, the present invention provides recombinantexpression constructs capable of directing the expression of constructsof the invention, including binding domain-immunoglobulin fusionproteins, as provided herein. The amino acids, which occur in thevarious amino acid sequences referred to herein, are identifiedaccording to their well known three-letter or single-letterabbreviations. The nucleotides, which occur in the various DNA sequencesor fragments thereof referred herein, are designated with the standardsingle letter designations used routinely in the art. A given amino acidsequence may also encompass similar but changed amino acid sequences,such as those having only minor changes, for example by way ofillustration and not limitation, covalent chemical modifications,insertions, deletions and substitutions, which may further includeconservative substitutions or substitutions with non-naturally-occurringamino acids. Amino acid sequences that are similar to one another mayshare substantial regions of sequence homology. In like fashion,nucleotide sequences may encompass substantially similar nucleotidesequences having only minor changes, for example by way of illustrationand not limitation, covalent chemical modifications, insertions,deletions and substitutions, which may further include silent mutationsowing to degeneracy of the genetic code. Nucleotide sequences that aresimilar to one another may share substantial regions of sequencehomology.

The presence of a malignant condition in a subject refers to thepresence of dysplastic, cancerous, and/or transformed cells in thesubject, including, for example, neoplastic, tumor, non-contactinhibited, or oncogenically transformed cells, or the like (e.g.,melanoma, carcinomas such as adenocarcinoma, squamous cell carcinoma,small cell carcinoma, oat cell carcinoma, etc., sarcomas such aschondrosarcoma, osteosarcoma, etc.) which are known to the art and forwhich criteria for diagnosis and classification are established. Inpreferred embodiments contemplated by the present invention, forexample, such cancer cells are malignant hematopoietic cells, such astransformed cells of lymphoid lineage and in particular, B celllymphomas and the like; cancer cells may in certain preferredembodiments also be epithelial cells such as carcinoma cells. Theinvention also contemplates B cell disorders, which may include certainmalignant conditions that affect B cells (e.g., B cell lymphoma) butwhich is not intended to be so limited, and which is also intended toencompass autoimmune diseases and in particular, diseases, disorders andconditions that are characterized by autoantibody production, forexample.

Autoantibodies are antibodies that react with self-antigens.Autoantibodies are detected in several autoimmune diseases (i.e., adisease, disorder or condition wherein a host immune system generates aninappropriate anti-“self” immune reaction) where they are involved indisease activity. Current treatments for various autoimmune diseasesinclude immunosuppressive drugs that require continuing administration,lack specificity, and cause significant side effects. New approachesthat can eliminate autoantibody production with minimal toxicity willaddress an unmet medical need for a spectrum of diseases that affectmany people. Constructs of the subject invention, including bindingdomain-immunoglobulin fusion proteins, are designed, for example, forimproved penetration into lymphoid tissues. Depletion of B lymphocytesinterrupts the autoantibody production cycle, and allows the immunesystem to reset as new B lymphocytes are produced from precursors in thebone marrow.

A number of diseases, disorders, and conditions have been identified forwhich beneficial effects are believed, according to non-limiting theory,to result from B cell depletion therapy. Such diseases disorders, andconditions include, but are not limited to, Grave's disease, Hashimoto'sdisease, rheumatoid arthritis, systemic lupus erythematosus, SjogrensSyndrome Immune Thrombocytopenic purpura, multiple sclerosis, myastheniagravis, scleroderma, psoriasis, Inflamatory Bowel Disease includingCrohn's disease and ulcerative colitis. Inflamatory Bowel Diseaseincluding Crohn's disease and Ulcerative colitis, are autoimmunediseases of the digestive system.

The present invention further relates to nucleotide constructs encodingconstructs of the invention, for example, binding domain-immunoglobulinfusion proteins, and in particular to methods for administeringrecombinant constructs encoding such proteins for gene therapyapplications that may be expressed, for example, as fragments, analogsand derivatives of such polypeptides.

The terms “fragment,” “derivative” and “analog” when referring toconstructs of the invention including, for example, bindingdomain-immunoglobulin fusion polypeptides or fusion proteins, refers toany construct, such as a binding domain-immunoglobulin fusionpolypeptide or fusion protein, that retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a pro- or prepro-form of a construct, for example, apro-protein that can be activated by cleavage of the pro-protein portionto produce an active construct, such as a binding domain-immunoglobulinfusion polypeptide.

A fragment, derivative or analog of a construct of the invention, forexample, a binding domain-immunoglobulin fusion polypeptide or fusionprotein, including binding domain-immunoglobulin fusion polypeptides orfusion proteins encoded by the cDNAs referred to herein, may be (i) onein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich additional amino acids are fused or otherwise connected to theconstruct, e.g., a binding domain-immunoglobulin fusion polypeptide,including amino acids that are employed for detection or specificfunctional alteration of the construct, inlcuding such constructs as abinding domain-immunoglobulin fusion polypeptide or a proproteinsequence. Such fragments, derivatives and analogs are deemed to bewithin the scope of those skilled in the art from the teachings herein.

The constructs, including polypeptide constructs, of the presentinvention include, for example, binding domain-immunoglobulin fusionpolypeptides and fusion proteins having binding regions such as bindingdomain polypeptide amino acid sequences that are identical or similar tosequences known in the art, or fragments or portions thereof. Forexample by way of additional illustration and not limitation, a humanCD154 molecule extracellular domain is contemplated for use according tothe instant invention, as are portions of such polypeptides and/orpolypeptides having at least about 70% similarity (preferably greaterthan a 70% identity) and more preferably about 90% similarity (morepreferably greater than a 90% identity) to the reported polypeptide andstill more preferably about 95% similarity (still more preferablygreater than a 95% identity) to the reported polypeptides and toportions of such polypeptides, wherein such portions of a bindingdomain-immunoglobulin fusion polypeptide, for example, generally containat least about 30 amino acids and more preferably at least about 50amino acids. Extracellular domains include, for example, portions of acell surface molecule, and in particularly preferred embodiments cellsurface molecules that are integral membrane proteins or that comprise aplasma membrane spanning transmembrane domain, that are constructed toextend beyond the outer leaflet of the plasma membrane phospholipidbilayer when the molecule is expressed at a cell surface, preferably ina manner that exposes the extracellular domain portion of such amolecule to the external environment of the cell, also known as theextracellular milieu. Methods for determining whether a portion of acell surface molecule comprises an extracellular domain are well knownto the art and include, for example, experimental determination (e.g.,direct or indirect labeling of the molecule, evaluation of whether themolecule can be structurally altered by agents to which the plasmamembrane is not permeable such as proteolytic or lipolytic enzymes) ortopological prediction based on the structure of the molecule (e.g.,analysis of the amino acid sequence of a polypeptide) or othermethodologies.

As used herein, an “amino acid” is a molecule having the structurewherein a central carbon atom (the alpha (α)-carbon atom) is linked to ahydrogen atom, a carboxylic acid group (the carbon atom of which isreferred to herein as a “carboxylcarbon atom”), an amino group (thenitrogen atom of which is referred to herein as an “amino nitrogenatom”), and a side chain group, R. When incorporated into a peptide,polypeptide, or protein, an amino acid loses one or more atoms of itsamino and carboxylic groups in the dehydration reaction that links oneamino acid to another. As a result, when incorporated into a protein, anamino acid may also be referred to as an “amino acid residue.” In thecase of naturally occurring proteins, an amino acid residue's R groupdifferentiates the 20 amino acids from which proteins are typicallysynthesized, although one or more amino acid residues in a protein maybe derivatized or modified following incorporation into protein inbiological systems (e.g., by glycosylation and/or by the formation ofcystine through the oxidation of the thiol side chains of twonon-adjacent cysteine amino acid residues, resulting in a disulfidecovalent bond that frequently plays an important role in stabilizing thefolded conformation of a protein, etc.). As those in the art willappreciate, non-naturally occurring amino acids can also be incorporatedinto proteins, particularly those produced by synthetic methods,including solid state and other automated synthesis methods. Examples ofsuch amino acids include, without limitation, α-amino isobutyric acid,4-amino butyric acid, L-amino butyric acid, 6-amino hexanoic acid,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norlensine,norvaline, hydroxproline, sarcosine, citralline, cysteic acid,t-butylglyine, t-butylalanine, phenylylycine, cyclohexylalanine,13-alanine, fluoro-amino acids, designer amino acids (e.g., β-methylamino acids, α-methyl amino acids, Nα-methyl amino acids) and amino acidanalogs in general. In addition, when an α-carbon atom has fourdifferent groups (as is the case with the 20 amino acids used bybiological systems to synthesize proteins, except for glycine, which hastwo hydrogen atoms bonded to the α carbon atom), two differentenantiomeric forms of each amino acid exist, designated D and L. Inmammals, only L-amino acids are incorporated into naturally occurringpolypeptides. The instant invention envisions proteins incorporating oneor more D- and L-amino acids, as well as proteins comprised of just D-or L-amino acid residues.

Herein, the following abbreviations may be used for the following aminoacids (and residues thereof): alanine (Ala, A); arginine (Arg, R);asparagine (Asn, N); aspartic acid (Asp, D); cyteine (Cys, C); glycine(Gly, G); glutamic acid (Glu, E); glutamine (Gln, Q); histidine (His,H); isoleucine (Ile, I); leucine (Leu, L); lysine (Lys, K); methionine(Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S);threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine(Val, V). Non-polar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionines.Neutral amino acids include glycine, serine, threonine, cysteine,tyrosine, esparagine, and glutamine. Positively charged (basic aminoacids include arginine, lysine and histidine. Negatively charged(acidic) amino acids include aspartic acid and glutamic acid.

“Protein” refers to any polymer of two or more individual amino acids(whether or not naturally occurring) linked via a peptide bond, andoccurs when the carboxylcarbon atom of the carboxylic acid group bondedto the α-carbon of one amino acid (or amino acid residue) becomescovalently bound to the amino nitrogen atom of amino group bonded to theα-carbon of an adjacent amino acid. The term “protein” is understood toinclude the terms “polypeptide” and “peptide” (which, at times, may beused interchangeably herein) within its meaning In addition, proteinscomprising multiple polypeptide subunits or other components will alsobe understood to be included within the meaning of “protein” as usedherein. Similarly, fragments of proteins, peptides, and polypeptides arealso within the scope of the invention and may be referred to herein as“proteins.”

In biological systems (be they in vivo or in vitro, including cell-free,systems), the particular amino acid sequence of a given protein (i.e.,the polypeptide's “primary structure,” when written from theamino-terminus to carboxy-terminus) is determined by the nucleotidesequence of the coding portion of a mRNA, which is in turn specified bygenetic information, typically genomic DNA (which, for purposes of thisinvention, is understood to include organelle DNA, for example,mitochondrial DNA and chloroplast DNA). Of course, any type of nucleicacid which constitutes the genome of a particular organism (e.g.,double-stranded DNA in the case of most animals and plants, single ordouble-stranded RNA in the case of some viruses, etc.) is understood tocode for the gene product(s) of the particular organism. Messenger RNAis translated on a ribosome, which catalyzes the polymerization of afree amino acid, the particular identity of which is specified by theparticular codon (with respect to mRNA, three adjacent A, G, C, or Uribonucleotides in the mRNA's coding region) of the mRNA then beingtranslated, to a nascent polypeptide. Recombinant DNA techniques haveenabled the large-scale synthesis of proteins and polypeptides (e.g.,human insulin, human growth hormone, erythropoietin, granulocyte colonystimulating factor, etc.) having the same primary sequence as whenproduced naturally in living organisms. In addition, such technology hasallowed the synthesis of analogs of these and other proteins, whichanalogs may contain one or more amino acid deletions, insertions, and/orsubstitutions as compared to the native proteins. Recombinant DNAtechnology also enables the synthesis of entirely novel proteins.

In non-biological systems (e.g., those employing solid state synthesis),the primary structure of a protein (which also includes disulfide(cystine) bond locations) can be determined by the user. As a result,polypeptides having a primary structure that duplicates that of abiologically produced protein can be achieved, as can analogs of suchproteins. In addition, completely novel polypeptides can also besynthesized, as can protein incorporating non-naturally occurring aminoacids.

As is known in the art, “similarity” between two polypeptides may bedetermined by comparing the amino acid sequence (including conservedamino acid substitutes therein) of one polypeptide to the sequence of asecond polypeptide. Fragments or portions of the nucleic acids encodingpolypeptides of the present invention may be used to synthesizefull-length nucleic acids of the present invention. As used herein, “%identity” refers to the percentage of identical amino acids situated atcorresponding amino acid residue positions when two or more polypeptideare aligned and their sequences analyzed using, for example, a gappedBLAST algorithm (e.g., Altschul et al., 1997 Nucl. Ac. Res. 25:3389;Altschul et al., 1990 J. Mol. Biol., 215:403-410) which weights sequencegaps and sequence mismatches according to the default weightingsprovided by the National Institutes of Health/NCBI database (Bethesda,Md.; see www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast). Otheralignment methods include BLITZ (MPsrch) (Sturrock & Collins, 1993), andFASTA (Pearson and Lipman, 1988 Proc. Natl. Acad. Sci. USA.85:2444-2448).

The term “isolated” means, in the case of a naturally occurringmaterial, that the material is or has been removed from, or is no longerassociated with, its natural or original environment. For example, anaturally occurring nucleic acid or protein or polypeptide present in aliving animal is not isolated, but the same nucleic acid or polypeptide,separated from some or all of the co-existing materials in the naturalsystem, is isolated. Such nucleic acids could be part of a vector and/orsuch nucleic acids or polypeptides could be part of a composition, andstill be isolated in that such vector or composition is not part of itsnatural environment. The term “isolated”, in the case of non-naturallyoccurring material, such as a recombinantly manufactured construct ofthe invention, includes material that is substantially or essentiallyfree from components which normally accompany it during manufacture,such as, for example, proteins and peptides that have been purified to adesired degree, preferably, for example, so that they are at least about80% pure, more preferably at least about 90%, and still more preferablyat least about 95% as measured by techniques known in the art.

The term “gene” means a segment of DNA involved in producing apolypeptide chain; it may also include regions preceding and following apolypeptide coding region, for example, a “leader and trailer” as wellas intervening sequences (introns) between relevant individual codingsegments (exons).

As described herein, the invention provides constructs, includingbinding domain-immunoglobulin fusion proteins, that may be encoded inwhole or in part by nucleic acids that have a binding region codingsequence such as, for example, a binding domain coding sequence fused orotherwise connected in frame to an additional native or engineeredimmunoglobulin domain encoding sequence to provide for expression of,for example, a binding domain polypeptide sequence fused or otherwiseconnected to an additional functional polypeptide sequence that permits,for example by way of illustration and not limitation, detection,functional alteration, isolation and/or purification of the fusionprotein. Such fusion proteins may permit functional alteration of abinding domain by containing additional immunoglobulin-derivedpolypeptide sequences that influence behavior of the fusion product, forexample (and as described above) by reducing the availability ofsufhydryl groups for participation in disulfide bond formation, and byconferring the ability to potentiate ADCC and/or CDC and/or fixcomplement.

Modification of a polypeptide may be effected by any means known tothose of skill in this art. The preferred methods herein rely onmodification of DNA encoding, for example, a fusion protein andexpression of the modified DNA. DNA encoding one of the constructs ofthe invention, for example, one of the binding domain-immunoglobulinfusions discussed herein, for example, may be altered or mutagenizedusing standard methodologies, including those described below. Forexample, cysteine residues that may otherwise facilitate multimerformation or promote particular molecular conformations can be deletedfrom a polypeptide or replaced, e.g., cysteine residues that areresponsible for or participate in aggregate formation. If necessary, forexample, the identity of cysteine residues that contribute to aggregateformation may be determined empirically, by deleting and/or replacing acysteine residue and ascertaining whether the resulting proteinaggregates in solutions containing physiologically acceptable buffersand salts. In addition, fragments of, for example, bindingdomain-immunoglobulin fusions may be constructed and used. As notedabove, counterreceptor/ligand binding domains for many candidate bindingdomain-immunoglobulin fusion have been delineated, such that one havingordinary skill in the art may readily select appropriate polypeptidedomains for inclusion in encoded products of the instant expressionconstructs.

Conservative substitutions of amino acids are well known and may be madegenerally without altering the biological activity of the resultingbinding domain-immunoglobulin fusion protein molecule. For example, suchsubstitutions are generally made by interchanging within the groups ofpolar residues, charged residues, hydrophobic residues, small residues,and the like. If necessary, such substitutions may be determinedempirically merely by testing the resulting modified protein for theability to bind to the appropriate cell surface receptors in in vitrobiological assays, or to bind to appropriate antigens or desired targetmolecules.

The present invention further relates to nucleic acids which hybridizeto constructs of the invention, including for example, bindingdomain-immunoglobulin fusion protein encoding polynucleotide sequencesas provided herein, or their complements, as will be readily apparent tothose familiar with the art, if there is at least about 70%, preferablyat least about 80-85%, more preferably at least about 90%, and stillmore preferably at least about 95%, 96%, 97%, 98% or 99% identitybetween the sequences.

The present invention particularly relates to nucleic acids thathybridize under stringent conditions to, for example, the bindingdomain-immunoglobulin fusion encoding nucleic acids referred to herein.As used herein, to “hybridize” under conditions of a specifiedstringency is used to describe the stability of hybrids formed betweentwo single-stranded nucleic acid molecules. Stringency of hybridizationis typically expressed in conditions of ionic strength and temperatureat which such hybrids are annealed and washed. The term “stringentconditions” refers to conditions that permit hybridization betweenpolynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent (e.g., formamide),temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of organic solvents (e.g., formamide), orraising the hybridization temperature. For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and most preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of an organic solvent(e.g., at least about 35% formamide, most preferably at least about 50%formamide). Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. Varying additionalparameters, for example, hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed, and are within the skill in the art. Other typical“high”, “medium” and “low” stringency encompass the following conditionsor equivalent conditions thereto: high stringency: 0.1×SSPE or SSC, 0.1%SDS, 65° C.; medium stringency: 0.2×SSPE or SSC, 0.1% SDS, 50° C.; andlow stringency: 1.0×SSPE or SSC, 0.1% SDS, 50° C. As known to thosehaving ordinary skill in the art, variations in stringency ofhybridization conditions may be achieved by altering the time,temperature and/or concentration of the solutions used forprehybridization, hybridization and wash steps, and suitable conditionsmay also depend in part on the particular nucleotide sequences of theprobe used, and of the blotted, proband nucleic acid sample.Accordingly, it will be appreciated that suitably stringent conditionscan be readily selected without undue experimentation where a desiredselectivity of the probe is identified, based on its ability tohybridize to one or more certain proband sequences while not hybridizingto certain other proband sequences.

As used herein, preferred “stringent conditions” generally refer tohybridization that will occur only if there is at least about 90-95% andmore preferably at least about 97% identity between the sequences. Thenucleic acid constructs which hybridize to, for example, bindingdomain-immunoglobulin fusion encoding nucleic acids referred to herein,in preferred embodiments, encode polypeptides which retain substantiallythe same biological function or activity as, for example, the bindingdomain-immunoglobulin fusion polypeptides encoded by the cDNAs.

The nucleic acids of the present invention, also referred to herein aspolynucleotides, may be in the form of RNA, for example, mRNA, or in theform of DNA, which DNA includes cDNA (also called “complementary DNA”,which is a DNA molecule that is complementary to a specific messengerRNA), genomic DNA, and synthetic DNA. The DNA may be double-stranded orsingle-stranded, and if single stranded may be the coding strand ornon-coding (anti-sense) strand. A coding sequence which encodes aconstruct of the invention, for example, a binding domain-immunoglobulinfusion polypeptide for use according to the invention may containportions that are identical to the coding sequence known in the art ordescribed herein for portions thereof, or may be a different codingsequence, which, as a result of the redundancy or degeneracy of thegenetic code, encodes the same construct or portion thereof, includingall or a portion of a binding domain-immunoglobulin fusion polypeptide.

The nucleic acids which encode constructs of the invention, for example,binding domain-immunoglobulin fusion polypeptides, for use according tothe invention may include, but are not limited to: only the codingsequence for the construct, such as a binding domain-immunoglobulinfusion polypeptide; the coding sequence for the construct, such as abinding domain-immunoglobulin fusion polypeptide and additional codingsequence; the coding sequence for the construct, such as a bindingdomain-immunoglobulin fusion polypeptide (and optionally additionalcoding sequence) and non-coding sequence, such as introns or non-codingsequences 5′ and/or 3′ of the coding sequence for the bindingdomain-immunoglobulin fusion polypeptide or a portion(s) thereof, whichfor example may further include but need not be limited to one or moreregulatory nucleic acid sequences that may be a regulated or regulatablepromoter, enhancer, other transcription regulatory sequence, repressorbinding sequence, translation regulatory sequence or any otherregulatory nucleic acid sequence. Thus, the term “nucleic acid encoding”or “polynucleotide encoding” a construct, for example, a bindingdomain-immunoglobulin fusion protein, encompasses a nucleic acid whichincludes only coding sequence for, for example, a bindingdomain-immunoglobulin fusion polypeptide as well as a nucleic acid whichincludes additional coding and/or non-coding sequence(s).

Nucleic acids and oligonucleotides for use as described herein can besynthesized by any method known to those of skill in this art (see,e.g., WO 93/01286, U.S. application Ser. No. 07/723,454; U.S. Pat. No.5,218,088; U.S. Pat. No. 5,175,269; U.S. Pat. No. 5,109,124).Identification of various oligonucleotides and nucleic acid sequencesalso involves methods known in the art. For example, the desirableproperties, lengths and other characteristics of oligonucleotides usefulfor cloning are well known. In certain embodiments, syntheticoligonucleotides and nucleic acid sequences may be designed that resistdegradation by endogenous host cell nucleolytic enzymes by containingsuch linkages as: phosphorothioate, methylphosphonate, sulfone, sulfate,ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and othersuch linkages that have proven useful in antisense applications. See,e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542 (1987); Miller etal., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec et al., TetrehedronLett. 26:2191-2194 (1985); Moody et al., Nucl. Acids Res. 12:4769-4782(1989); Uznanski et al., Nucl. Acids Res. (1989); Letsinger et al.,Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev. Biochem. 54:367-402(1985); Eckstein, Trends Biol. Sci. 14:97-100 (1989); Stein In:Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen,Ed, Macmillan Press, London, pp. 97-117 (1989); Jager et al.,Biochemistry 27:7237-7246 (1988).

In one embodiment, the present invention provides truncated components(e.g., binding domain polypeptide, hinge region polypeptide, linker,etc.) for use in a construct of the invention, for example, a bindingdomain-immunoglobulin fusion protein. In another embodiment theinvention provides nucleic acids encoding a construct of the invention,for example, a binding domain-immunoglobulin fusion protein having suchtruncated components. A truncated molecule may be any molecule thatcomprises less than a full length version of the molecule of interest.Truncated molecules provided by the present invention may includetruncated biological polymers, and in preferred embodiments of theinvention such truncated molecules may be truncated nucleic acidmolecules or truncated polypeptides. Truncated nucleic acid moleculeshave less than the full length nucleotide sequence of a known ordescribed nucleic acid molecule, where such a known or described nucleicacid molecule may be a naturally occurring, a synthetic, or arecombinant nucleic acid molecule, so long as one skilled in the artwould regard it as a full length molecule. Thus, for example, truncatednucleic acid molecules that correspond to a gene sequence contain lessthan the full length gene where the gene comprises coding and non-codingsequences, promoters, enhancers and other regulatory sequences, flankingsequences and the like, and other functional and non-functionalsequences that are recognized as part of the gene. In another example,truncated nucleic acid molecules that correspond to a mRNA sequencecontain less than the full length mRNA transcript, which may includevarious translated and non-translated regions as well as otherfunctional and non-functional sequences.

In other preferred embodiments, truncated molecules are polypeptidesthat comprise less than the full-length amino acid sequence of aparticular protein or polypeptide component. As used herein “deletion”has its common meaning as understood by those familiar with the art, andmay refer to molecules that lack one or more portions of a sequence fromeither terminus or from a non-terminal region, relative to acorresponding full length molecule, for example, as in the case oftruncated molecules provided herein. Truncated molecules that are linearbiological polymers such as nucleic acid molecules or polypeptides mayhave one or more of a deletion from either terminus of the moleculeand/or one or more deletions from a non-terminal region of the molecule,where such deletions may be deletions of from about 1-1500 contiguousnucleotide or amino acid residues, preferably about 1-500 contiguousnucleotide or amino acid residues and more preferably about 1-300contiguous nucleotide or amino acid residues, including deletions ofabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-40, 41-50, 51-74, 75-100,101-150, 151-200, 201-250 or 251-299 contiguous nucleotide or amino acidresidues. In certain particularly preferred embodiments truncatednucleic acid molecules may have a deletion of about 270-330 contiguousnucleotides. In certain more preferred embodiments, truncatedpolypeptide molecules may have a deletion, for example, of about 80-140contiguous amino acids.

The present invention further relates to variants of the hereinreferenced nucleic acids that encode fragments, analogs and/orderivatives of a construct of the invention, for example, a bindingdomain-immunoglobulin fusion polypeptide. The variants of the nucleicacids encoding constructs of the invention, for example, bindingdomain-immunoglobulin fusion proteins, may be naturally occurringallelic variants of one or more portions of the nucleic acid sequencesincluded therein, or non-naturally occurring variants of such sequencesor portions or sequences, including sequences varied by molecularengineering using, for example, methods know in the art for varyingsequence. As is known in the art, an allelic variant is an alternateform of a nucleic acid sequence which may have at least one of asubstitution, a deletion or an addition of one or more nucleotides, anyof which does not substantially or undesireably alter the function ofthe encoded binding domain-immunoglobulin fusion polypeptide.

Variants and derivatives of constructs of the invention, for example,binding domain-immunoglobulin fusion proteins, may be obtained bymutations of nucleotide sequences encoding, for example, bindingdomain-immunoglobulin fusion polypeptides or any portion thereof.Alterations of the native amino acid sequence may be accomplished by anyof a number of conventional methods. Mutations can be introduced atparticular loci, for example, by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes an analog having the desiredamino acid insertion, substitution, or deletion.

Alternatively, for example, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered genewherein predetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making such alterations are disclosed byWalder et al., 1986 Gene 42:133; Bauer et al., 1985 Gene 37:73; Craik,January 1985 BioTechniques 12-19; Smith et al., January 1985 GeneticEngineering: Principles and Methods BioTechniques 12-19; Costa G L, etal., “Site-directed mutagenesis using a rapid PCR-based method,” 1996Methods Mol. Biol. 57:239-48; Rashtchian A., “Novel methods for cloningand engineering genes using the polymerase chain reaction,” 1995 CurrOpin Biotechnol. 6(1):30-6; Sharon J, et al., “Oligonucleotide-directedmutagenesis of antibody combining sites,” 1993 Int Rev Immunol.10(2-3):113-27; Kunkel, 1985 Proc. Natl. Acad. Sci. USA 82:488; Kunkelet al., 1987 Methods in Enzymol. 154:367; and, U.S. Pat. Nos. 4,518,584and 4,737,462.

As an example, modification of DNA may be performed by site-directedmutagenesis of DNA encoding a protein combined with the use of DNAamplification methods using primers to introduce and amplify alterationsin the DNA template, such as PCR splicing by overlap extension (SOE).Site-directed mutagenesis is typically effected using a phage vectorthat has single- and double-stranded forms, such as M13 phage vectors,which are well known and commercially available. Other suitable vectorsthat contain a single-stranded phage origin of replication may be used.See, e.g., Veira et al., 1987 Meth. Enzymol. 15:3. In general,site-directed mutagenesis is performed by preparing a single-strandedvector that encodes the protein of interest (e.g., all or a componentportion of a given binding domain-immunoglobulin fusion protein). Anoligonucleotide primer that contains the desired mutation within aregion of homology to the DNA in the single-stranded vector is annealedto the vector followed by addition of a DNA polymerase, such as E. coliDNA polymerase I (Klenow fragment), which uses the double strandedregion as a primer to produce a heteroduplex in which one strand encodesthe altered sequence and the other the original sequence. Theheteroduplex is introduced into appropriate bacterial cells and clonesthat include the desired mutation are selected. The resulting alteredDNA molecules may be expressed recombinantly in appropriate host cellsto produce the modified protein.

Equivalent DNA constructs that include code for additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences not needed or desired forbiological activity, for example, are also encompassed by the invention.For example, and as discussed above, sequences encoding Cys residuesthat are not desirable or essential for biological activity can bealtered to cause the Cys residues to be deleted or replaced with otheramino acids, for example, thus preventing formation of incorrect orundesired intramolecular disulfide bridges upon synthesis orrenaturation.

A “host cell” or “recombinant host cell” is a cell that contains avector, e.g., an expression vector, or a cell that has otherwise beenmanipulated by recombinant techniques to express a protein of interest.Host organisms include those organisms in which recombinant productionof constructs of the invention, for example, bindingdomain-immunoglobulin fusion products encoded by the recombinantconstructs of the present invention may occur, such as bacteria (forexample, E. coli), yeast (for example, Saccharomyces cerevisiae andPichia pastoris), insect cells, and mammalian cells, including in vitroand in vivo expression. Host organisms thus may include organisms forthe construction, propagation, expression or other steps in theproduction of the compositions provided herein. Hosts include subjectsin which immune responses take place, as described herein. Presentlypreferred host organisms for production of constructs of the inventionthat produce glycosylated proteins are mammalian cells or other cellssystems that pemit the expression and recovery of glycosylated proteins.Other cell lines include inbred murine strains and murine cell lines,and human cellsand cell lines.

A DNA construct encoding a desired construct of the invention, forexample, a binding domain-immunoglobulin fusion protein is introducedinto a vector, for example, a plasmid, for expression in an appropriatehost. In preferred embodiments, the host is a mammalian host, forexample, a mammalian cell line. The sequence encoding the ligand ornucleic acid binding domain is preferably codon-optimized for expressionin the particular host. Thus, for example, if a construct, for example,is a human binding domain-immunoglobulin fusion and is expressed inbacteria, the codons may be optimized for bacterial usage. For smallcoding regions, the gene can be synthesized as a single oligonucleotide.For larger proteins, splicing of multiple oligonucleotides, mutagenesis,or other techniques known to those in the art may be used. The sequencesof nucleotides in plasmids or other vectors that are regulatory regions,such as promoters and operators, are operationally associated with oneanother for transcription. The sequence of nucleotides encoding abinding domain-immunoglobulin fusion protein may also include DNAencoding a secretion signal, whereby the resulting peptide is aprecursor protein. The resulting processed protein may be recovered fromthe periplasmic space or the fermentation medium.

In preferred embodiments, the DNA plasmids may also include atranscription terminator sequence. As used herein, a “transcriptionterminator region” is a sequence that signals transcription termination.The entire transcription terminator may be obtained from aprotein-encoding gene, which may be the same or different from theinserted binding domain-immunoglobulin fusion encoding gene or thesource of the promoter. Transcription terminators are optionalcomponents of the expression systems herein, but are employed inpreferred embodiments.

The plasmids or other vectors used herein include a promoter inoperative association with the DNA encoding the protein or polypeptideof interest and are designed for expression of proteins in a suitablehost as described above (e.g., bacterial, murine, or human) dependingupon the desired use of the plasmid (e.g., administration of a vaccinecontaining binding domain-immunoglobulin fusion encoding sequences).Suitable promoters for expression of proteins and polypeptides hereinare widely available and are well known in the art. Inducible promotersor constitutive promoters that are linked to regulatory regions arepreferred. Such promoters include, for example, but are not limited to,the T7 phage promoter and other T7-like phage promoters, such as the T3,T5 and SP6 promoters, the trp, lpp, and lac promoters, such as thelacUV5, from E. coli; the P10 or polyhedrin gene promoter ofbaculovirus/insect cell expression systems (see, e.g., U.S. Pat. Nos.5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784) and induciblepromoters from other eukaryotic expression systems. For expression ofthe proteins such promoters are inserted in a plasmid in operativelinkage with a control region such as the lac operon.

Preferred promoter regions are those that are inducible and functionalin mammalian cells, for example. Examples of suitable induciblepromoters and promoter regions for bacterial expression include, but arenot limited to: the E. coli lac operator responsive to isopropylβ-D-thiogalactopyranoside (IPTG; see Nakamura et al., 1979 Cell18:1109-1117); the metallothionein promoter metal-regulatory-elementsresponsive to heavy-metal (e.g., zinc) induction (see, e.g., U.S. Pat.No. 4,870,009); the phage T7lac promoter responsive to IPTG (see, e.g.,U.S. Pat. No. 4,952,496; and Studier et al., 1990 Meth. Enzymol.185:60-89) and the TAC promoter. Depending on the expression host systemto be used, plasmids may optionally include a selectable marker gene orgenes that are functional in the host. Thus, for example, a selectablemarker gene includes any gene that confers a phenotype on bacteria thatallows transformed bacterial cells to be identified and selectivelygrown from among a vast majority of untransformed cells. Suitableselectable marker genes for bacterial hosts, for example, include theampicillin resistance gene (Amp^(r)), tetracycline resistance gene(Tc^(r)) and the kanamycin resistance gene (Kan^(r)). The kanamycinresistance gene is presently preferred for bacterial expression.

In various expression systems, plasmids or other vectors may alsoinclude DNA encoding a signal for secretion of the operably linkedprotein. Secretion signals suitable for use are widely available and arewell known in the art. Prokaryotic and eukaryotic secretion signalsfunctional in E. coli may be employed. Depending on the expressionsystems, presently preferred secretion signals may include, but are notlimited to, those encoded by the following E. coli genes: ompA, ompT,ompF, ompC, beta-lactamase, and alkaline phosphatase, and the like (vonHeijne, J. Mol. Biol. 184:99-105, 1985). In addition, the bacterial pelBgene secretion signal (Lei et al., J. Bacteriol. 169:4379, 1987), thephoA secretion signal, and the cek2 functional in insect cell may beemployed. The most preferred secretion signal for certain expressionsystems is the E. coli ompA secretion signal. Other prokaryotic andeukaryotic secretion signals known to those of skill in the art may alsobe employed (see, e.g., von Heijne, J. Mol. Biol. 184:99-105, 1985).Using the methods described herein, one of skill in the art cansubstitute secretion signals that are functional, for example, in yeast,insect or mammalian cells to secrete proteins from those cells.

Preferred plasmids for transformation of E. coli cells include the pETexpression vectors (e.g., pET-11a, pET-12a-c, pET-15b; see U.S. Pat. No.4,952,496; available from Novagen, Madison, Wis.). Other preferredplasmids include the pKK plasmids, particularly pKK 223-3, whichcontains the tac promoter (Brosius et al., 1984 Proc. Natl. Acad. Sci.81:6929; Ausubel et al., Current Protocols in Molecular Biology; U.S.Pat. Nos. 5,122,463, 5,173,403, 5,187,153, 5,204,254, 5,212,058,5,212,286, 5,215,907, 5,220,013, 5,223,483, and 5,229,279). Plasmid pKKhas been modified by replacement of the ampicillin resistance gene witha kanamycin resistance gene. (Available from Pharmacia; obtained frompUC4K, see, e.g., Vieira et al. (1982 Gene 19:259-268; and U.S. Pat. No.4,719,179.) Baculovirus vectors, such as pBlueBac (also called pJVETLand derivatives thereof), particularly pBlueBac III (see, e.g., U.S.Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317,4,745,051, and 5,169,784; available from Invitrogen, San Diego) may alsobe used for expression of the polypeptides in insect cells. Otherplasmids include the pIN-IIIompA plasmids (see U.S. Pat. No. 4,575,013;see also Duffaud et al., Meth. Enz. 153:492-507, 1987), such aspIN-IIIompA2.

Preferably, if one or more DNA molecules is replicated in bacterialcells, the preferred host is E. coli. The preferred DNA molecule is sucha system also includes a bacterial origin of replication, to ensure themaintenance of the DNA molecule from generation to generation of thebacteria. In this way, large quantities of the DNA molecule can beproduced by replication in bacteria. In such expression systems,preferred bacterial origins of replication include, but are not limitedto, the fl-ori and col E1 origins of replication. Preferred hosts forsuch systems contain chromosomal copies of DNA encoding T7 RNApolymerase operably linked to an inducible promoter, such as the lacUVpromoter (see U.S. Pat. No. 4,952,496). Such hosts include, but are notlimited to, lysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS,HMS174(DE3) and BL21(DE3). Strain BL21(DE3) is preferred. The pLysstrains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNApolymerase.

The DNA molecules provided may also contain a gene coding for arepressor protein. The repressor protein is capable of repressing thetranscription of a promoter that contains sequences of nucleotides towhich the repressor protein binds. The promoter can be derepressed byaltering the physiological conditions of the cell. For example, thealteration can be accomplished by adding to the growth medium a moleculethat inhibits the ability to interact with the operator or withregulatory proteins or other regions of the DNA or by altering thetemperature of the growth media. Preferred repressor proteins include,but are not limited to the E. coli lad repressor responsive to IPTGinduction, the temperature sensitive λ cI857 repressor, and the like.The E. coli lad repressor is preferred.

In general, recombinant constructs of the subject invention will alsocontain elements necessary for transcription and translation. Inparticular, such elements are preferred where the recombinant expressionconstruct containing nucleic acid sequences encoding bindingdomain-immunoglobulin fusion proteins is intended for expression in ahost cell or organism. In certain embodiments of the present invention,cell type preferred or cell type specific expression of a cell bindingdomain-immunoglobulin fusion encoding gene may be achieved by placingthe gene under regulation of a promoter. The choice of the promoter willdepend upon the cell type to be transformed and the degree or type ofcontrol desired. Promoters can be constitutive or active and may furtherbe cell type specific, tissue specific, individual cell specific, eventspecific, temporally specific or inducible. Cell-type specific promotersand event type specific promoters are preferred. Examples ofconstitutive or nonspecific promoters include the SV40 early promoter(U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat. No.5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062), andadenovirus promoter. In addition to viral promoters, cellular promotersare also amenable within the context of this invention. In particular,cellular promoters for the so-called housekeeping genes are useful.Viral promoters are preferred, because generally they are strongerpromoters than cellular promoters. Promoter regions have been identifiedin the genes of many eukaryotes including higher eukaryotes, such thatsuitable promoters for use in a particular host can be readily selectedby those skilled in the art.

Inducible promoters may also be used. These promoters include MMTV LTR(PCT WO 91/13160), inducible by dexamethasone; metallothionein promoter,inducible by heavy metals; and promoters with cAMP response elements,inducible by cAMP. By using an inducible promoter, the nucleic acidsequence encoding a binding domain-immunoglobulin fusion protein may bedelivered to a cell by the subject invention expression construct andwill remain quiescent until the addition of the inducer. This allowsfurther control on the timing of production of the gene product.

Event-type specific promoters are active or up regulated only upon theoccurrence of an event, such as tumorigenicity or viral infection. TheHIV LTR is a well-known example of an event-specific promoter. Thepromoter is inactive unless the tat gene product is present, whichoccurs upon viral infection. Some event-type promoters are alsotissue-specific.

Additionally, promoters that are coordinately regulated with aparticular cellular gene may be used. For example, promoters of genesthat are coordinately expressed may be used when expression of aparticular binding construct of the invention, for example, a bindingdomain-immunoglobulin fusion protein-encoding gene is desired in concertwith expression of one or more additional endogenous or exogenouslyintroduced genes. This type of promoter is especially useful when oneknows the pattern of gene expression relevant to induction of an immuneresponse in a particular tissue of the immune system, so that specificimmunocompetent cells within that tissue may be activated or otherwiserecruited to participate in the immune response.

In addition to the promoter, repressor sequences, negative regulators,or tissue-specific silencers may be inserted to reduce non-specificexpression of binding domain-immunoglobulin fusion protein encodinggenes in certain situations, such as, for example, a host that istransiently immunocompromised as part of a therapeutic strategy.Multiple repressor elements may be inserted in the promoter region.Repression of transcription is independent on the orientation ofrepressor elements or distance from the promoter. One type of repressorsequence is an insulator sequence. Such sequences inhibit transcription(Dunaway et al., 1997 Mol Cell Biol 17: 182-9; Gdula et al., 1996 ProcNatl Acad Sci USA 93:9378-83, Chan et al., 1996 J Virol 70: 5312-28;Scott and Geyer, 1995 EMBO J. 14:6258-67; Kalos and Fournier, 1995 MolCell Biol 15:198-207; Chung et al., 1993 Cell 74: 505-14) and willsilence undesired background transcription.

Repressor elements have also been identified in the promoter regions ofthe genes for type II (cartilage) collagen, choline acetyltransferase,albumin (Hu et al., 1992 J. Cell Growth Differ. 3(9):577-588),phosphoglycerate kinase (PGK-2) (Misuno et al., 1992 Gene119(2):293-297), and in the6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene. (Lemaigre etal., Mol. Cell. Biol. 11(2):1099-1106). Furthermore, the negativeregulatory element Tse-1 has been identified in a number of liverspecific genes, and has been shown to block cAMP responseelement(CRE)-mediated induction of gene activation in hepatocytes.(Boshart et al., 1990 Cell 61(5):905-916,).

In preferred embodiments, elements that increase the expression of thedesired product are incorporated into the construct. Such elementsinclude internal ribosome binding sites (IRES; Wang and Siddiqui, 1995Curr. Top. Microbiol. Immunol 203:99; Ehrenfeld and Semler, 1995 Curr.Top. Microbiol. Immunol. 203:65; Rees et al., 1996 Biotechniques 20:102;Sugimoto et al., 1994 Biotechnology 12:694). IRES increase translationefficiency. As well, other sequences may enhance expression. For somegenes, sequences especially at the 5′ end inhibit transcription and/ortranslation. These sequences are usually palindromes that can formhairpin structures. Any such sequences in the nucleic acid to bedelivered are generally deleted. Expression levels of the transcript ortranslated product are assayed to confirm or ascertain which sequencesaffect expression. Transcript levels may be assayed by any known method,including Northern blot hybridization, RNase probe protection and thelike. Protein levels may be assayed by any known method, includingELISA, western blot, immunocytochemistry or other well-known techniques.

Other elements may be incorporated into the constructs of the invention,for example, into binding domain-immunoglobulin fusion protein encodingconstructs of the present invention. In preferred embodiments, theconstruct includes a transcription terminator sequence, including apolyadenylation sequence, splice donor and acceptor sites, and anenhancer. Other elements useful for expression and maintenance of theconstruct in mammalian cells or other eukaryotic cells may also beincorporated (e.g., origin of replication). Because the constructs areconveniently produced in bacterial cells, elements that are necessaryfor, or that enhance, propagation in bacteria are incorporated. Suchelements include an origin of replication, a selectable marker and thelike.

As provided herein, an additional level of controlling the expression ofnucleic acids encoding constructs of the invention, for example, bindingdomain-immunoglobulin fusion proteins, delivered to cells for genetherapy, for example, may be provided by simultaneously delivering twoor more differentially regulated nucleic acid constructs. The use ofsuch a multiple nucleic acid construct approach may permit coordinatedregulation of an immune response such as, for example, spatiotemporalcoordination that depends on the cell type and/or presence of anotherexpressed encoded component. Those familiar with the art will appreciatethat multiple levels of regulated gene expression may be achieved in asimilar manner by selection of suitable regulatory sequences, includingbut not limited to promoters, enhancers and other well known generegulatory elements.

The present invention also relates to vectors, and to constructsprepared from known vectors that include nucleic acids of the presentinvention, and in particular to “recombinant expression constructs”,including any of various known constructs, including deliveryconstructs, useful for gene therapy, that include any nucleic acidsencoding, for example, binding domain-immunoglobulin fusion proteins andpolypeptides according to the invention as provided herein; to hostcells which are genetically engineered with vectors and/or otherconstructs of the invention and to methods of administering expressionor other constructs comprising nucleic acid sequences encoding, forexample, binding domain-immunoglobulin fusion polypeptides and fusionproteins of the invention, or fragments or variants thereof, byrecombinant techniques.

Various constructs of the invention, including for example, bindingdomain-immunoglobulin fusion proteins, can be expressed in virtually anyhost cell, including in vivo host cells in the case of use for genetherapy, under the control of appropriate promoters, depending on thenature of the construct (e.g., type of promoter, as described above),and on the nature of the desired host cell (e.g., whether postmitoticterminally differentiated or actively dividing; e.g., whether theexpression construct occurs in host cell as an episome or is integratedinto host cell genome).

Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described, for example, by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989); as noted herein, in particularly preferredembodiments of the invention, recombinant expression is conducted inmammalian cells that have been transfected or transformed with thesubject invention recombinant expression construct. See also, forexample, Machida, Calif., “Viral Vectors for Gene Therapy Methods andProtocols”; Wolff, J A, “Gene Therapeutics: Methods and Applications ofDirect Gene Transfer” (Birkhauser 1994); Stein, U and Walther, W (eds.P, “Gene Therapy of Cancer: Methods and Protocols” (Humana Press 2000);Robbins, PD (ed.), “Gene Therapy Protocols” (Humana Press 1997); Morgan,J R (ed.), “Gene Therapy Protocols” (Humana Press 2002); Meager, A(ed.), “Gene Therapy Technologies, Applications and Regulations: FromLaboratory to Clinic” (John Wiley & Sons Inc. 1999); MacHida, C A andConstant, J G, “Viral Vectors for Gene Therapy: Methods and Protocols”(Humana Press 2002); “New Methods Of Gene Therapy For Genetic MetabolicDiseases NIH Guide,” Volume 22, Number 35, Oct. 1, 1993. See also recentU.S. patents relating to gene therapy, including vaccines, which includeU.S. Pat. Nos. 6,384,210 (“Solvent for biopolymer synthesis, solventmicrodroplets and methods of use”); 6,384,203 (“Family ofimmunoregulators designated leukocyte immunoglobulin-like receptors(LIR)”); 6,384,202 (“Cell-specific active compounds regulated by thecell cycle”); 6,384,018 (“Polynucleotide tuberculosis vaccine”);6,383,814 (“Cationic amphiphiles for intracellular delivery oftherapeutic molecules”); 6,383,811 (“Polyampholytes for deliveringpolyions to a cell”); 6,383,795 (“Efficient purification ofadenovirus”); 6,383,794 (“Methods of producing high titer recombinantadeno-associated virus”); 6,383,785 (“Self-enhancing, pharmacologicallycontrollable expression systems”); 6,383,753 (“Yeast mammalianregulators of cell proliferation”); 6,383,746 (“Functional promoter forCCR5”); 6,383,743 (“Method for serial analysis of gene expression”);6,383,738 (“Herpes simplex virus ORF P is a repressor of viral proteinsynthesis”); 6,383,737 (“Human oxalyl-CoA Decarboxylase”); 6,383,733(“Methods of screening for pharmacologically active compounds for thetreatment of tumour diseases”); 6,383,522 (“Toxicity reduced compositioncontaining an anti-neoplastic agent and a shark cartilage extract”);6,383,512 (“Vesicular complexes and methods of making and using thesame”); 6,383,481 (“Method for transplantation of hemopoietic stemcells”); 6,383,478 (“Polymeric encapsulation system promotingangiogenesis”); 6,383,138 (“Method for transdermal sampling ofanalytes”); 6,380,382 (“Gene encoding a protein having diagnostic,preventive, therapeutic, and other uses”); 6,380,371 (“Endoglycan: anovel protein having selectin ligand and chemokine presentationactivity”); 6,380,369 (“Human DNA mismatch repair proteins”); 6,380,362(“Polynucleotides, polypeptides expressed by the polynucleotides andmethods for their use”); 6,380,170 (“Nucleic acid construct for the cellcycle regulated expression of structural genes”); 6,380,169 (“Metalcomplex containing oligonucleoside cleavage compounds and therapies”);6,379,967 (“Herpesvirus saimiri as viral vector”); 6,379,966(“Intravascular delivery of non-viral nucleic acid protease proteins,and uses thereof”).

Typically, for example, expression constructs are derived from plasmidvectors. One preferred construct is a modified pNASS vector (Clontech,Palo Alto, Calif.), which has nucleic acid sequences encoding anampicillin resistance gene, a polyadenylation signal and a T7 promotersite. Other suitable mammalian expression vectors are well known (see,e.g., Ausubel et al., 1995; Sambrook et al., supra; see also, e.g.,catalogues from Invitrogen, San Diego, Calif.; Novagen, Madison, Wis.;Pharmacia, Piscataway, N.J.; and others). Presently preferred constructsmay be prepared that include a dihydrofolate reductase (DHFR) encodingsequence under suitable regulatory control, for promoting enhancedproduction levels of the binding domain-immunoglobulin fusion protei,which levels result from gene amplification following application of anappropriate selection agent (e.g., methetrexate).

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, as described above.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences. Thus, forexample, the binding domain-immunoglobulin fusion protein encodingnucleic acids as provided herein may be included in any one of a varietyof expression vector constructs as a recombinant expression constructfor expressing a binding domain-immunoglobulin fusion polypeptide in ahost cell. In certain preferred embodiments the constructs are includedin formulations that are administered in vivo. Such vectors andconstructs include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;yeast plasmids; vectors derived from combinations of plasmids and phageDNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, andpseudorabies, or replication deficient retroviruses as described below.However, any other vector may be used for preparation of a recombinantexpression construct, and in preferred embodiments such a vector will bereplicable and viable in the host.

The appropriate DNA sequence(s) may be inserted into a vector, forexample, by a variety of procedures. In general, a DNA sequence isinserted into an appropriate restriction endonuclease site(s) byprocedures known in the art. Standard techniques for cloning, DNAisolation, amplification and purification, for enzymatic reactionsinvolving DNA ligase, DNA polymerase, restriction endonucleases and thelike, and various separation techniques are those known and commonlyemployed by those skilled in the art. A number of standard techniquesare described, for example, in Ausubel et al. (1993 Current Protocols inMolecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc.,Boston, Mass.); Sambrook et al. (1989 Molecular Cloning, Second Ed.,Cold Spring Harbor Laboratory, Plainview, N.Y.); Maniatis et al. (1982Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.);Glover (Ed.) (1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK);Hames and Higgins (Eds.), (1985 Nucleic Acid Hybridization, IRL Press,Oxford, UK); and elsewhere.

The DNA sequence in the expression vector is operatively linked to atleast one appropriate expression control sequence(s) (e.g., aconstitutive promoter or a regulated promoter) to direct mRNA synthesis.Representative examples of such expression control sequences includepromoters of eukaryotic cells or their viruses, as described above.Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art, and preparation ofcertain particularly preferred recombinant expression constructscomprising at least one promoter or regulated promoter operably linkedto a nucleic acid encoding an binding domain-immunoglobulin fusionpolypeptide is described herein.

Transcription of the DNA encoding proteins and polypeptides includedwithin the present invention by higher eukaryotes may be increased byinserting an enhancer sequence into the vector. Enhancers are cis-actingelements of DNA, usually about from 10 to 300 by that act on a promoterto increase its transcription. Examples including the SV40 enhancer onthe late side of the replication origin by 100 to 270, a cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

Gene therapy is the use of genetic material to treat disease. Itcomprises strategies to replace defective genes or add new genes tocells and/or tissues, and is being developed for application in thetreatment of cancer, the correction of metabolic disorders and in thefield of immunotherapy. Gene therapies of the invention include the useof various constructs of the invention, with or without a separatecarrier or delivery vehicle or constructs, for treatment of thediseases, disorders, and/or conditions noted herein. Such constructs mayalso be used as vaccines for treatment or prevention of the diseases,disorders, and/or conditions noted herein. DNA vaccines, for example,make use of polynucleotides encoding immunogenic protein and nucleicacid determinants to stimulate the immune system against pathogens ortumor cells. Such strategies can stimulate either acquired or innateimmunity or can involve the modification of immune function throughcytokine expression. In vivo gene therapy involves the direct injectionof genetic material into a patient or animal model of human disease.Vaccines and immune modulation are systemic therapies. Withtissue-specific in vivo therapies, such as those that aim to treatcancer, localized gene delivery and/or expression/targeting systems arepreferred. Diverse gene therapy vectors have been designed to targetspecific tissues, and procedures have been developed to physicallytarget specific tissues, for example, using catheter-based technologies,all of which are contemplated herein. Ex vivo approaches to gene therapyare also contemplated herein and involve the removal, geneticmodification, expansion and re-administration of a patient's own cells.Examples include bone marrow transplantation for cancer treatment or thegenetic modivation of lymphoid progenitor cells. Ex vivo gene therapy ispreferably applied to the treatment of cells that are easily accessibleand can survive in culture during the gene transfer process (such asblood or skin cells).

Useful gene therapy vectors include adenoviral vectors, lentiviralvectors, Adeno-associated virus (AAV) vectors, Herpes Simplex Virus(Hsv) vectors, and retroviral vectors. Gene therapies may also becarried out using “naked DNA,” lipsome-based delivery, lipid-baseddelivery (including DNA attached to positively charged lipids), andelectroporation.

As provided herein, in certain embodiments, including but not limited togene therapy embodiments, the vector may be a viral vector such as, forexample, a retroviral vector. Miller et al., 1989 BioTechniques 7:980;Coffin and Varmus, 1996 Retroviruses, Cold Spring Harbor LaboratoryPress, NY. For example, retroviruses from which the retroviral plasmidvectors may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus.

Retroviruses are RNA viruses that can replicate and integrate into thegenome of a host cell via a DNA intermediate. This DNA intermediate, orprovirus, may be stably integrated into the host cell DNA. According tocertain embodiments of the present invention, an expression constructmay comprise a retrovirus into which a foreign gene that encodes aforeign protein is incorporated in place of normal retroviral RNA. Whenretroviral RNA enters a host cell coincident with infection, the foreigngene is also introduced into the cell, and may then be integrated intohost cell DNA as if it were part of the retroviral genome. Expression ofthis foreign gene within the host results in expression of the foreignprotein.

Most retroviral vector systems that have been developed for gene therapyare based on murine retroviruses. Such retroviruses exist in two forms,as free viral particles referred to as virions, or as provirusesintegrated into host cell DNA. The virion form of the virus contains thestructural and enzymatic proteins of the retrovirus (including theenzyme reverse transcriptase), two RNA copies of the viral genome, andportions of the source cell plasma membrane containing viral envelopeglycoprotein. The retroviral genome is organized into four main regions:the Long Terminal Repeat (LTR), which contains cis-acting elementsnecessary for the initiation and termination of transcription and issituated both 5′ and 3′ of the coding genes, and the three coding genesgag, pol, and env. These three genes gag, pol, and env encode,respectively, internal viral structures, enzymatic proteins (such asintegrase), and the envelope glycoprotein (designated gp70 and p15e)which confers infectivity and host range specificity of the virus, aswell as the “R” peptide of undetermined function.

Separate packaging cell lines and vector producing cell lines have beendeveloped because of safety concerns regarding the uses of retroviruses,including their use in expression constructs as provided by the presentinvention. Briefly, this methodology employs the use of two components,a retroviral vector and a packaging cell line (PCL). The retroviralvector contains long terminal repeats (LTRs), the foreign DNA to betransferred and a packaging sequence (y). This retroviral vector willnot reproduce by itself because the genes that encode structural andenvelope proteins are not included within the vector genome. The PCLcontains genes encoding the gag, pol, and env proteins, but does notcontain the packaging signal “y”. Thus, a PCL can only form empty virionparticles by itself. Within this general method, the retroviral vectoris introduced into the PCL, thereby creating a vector-producing cellline (VCL). This VCL manufactures virion particles containing only theretroviral vector's (foreign) genome, and therefore has previously beenconsidered to be a safe retrovirus vector for therapeutic use.

“Retroviral vector construct” refers to an assembly that is, withinpreferred embodiments of the invention, capable of directing theexpression of a sequence(s) or gene(s) of interest, such as bindingdomain-immunoglobulin fusion encoding nucleic acid sequences. Briefly,the retroviral vector construct must include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second strand DNA synthesis and a3′ LTR. A wide variety of heterologous sequences may be included withinthe vector construct, including for example, sequences which encode aprotein (e.g., cytotoxic protein, disease-associated antigen, immuneaccessory molecule, or replacement gene), or which are useful as amolecule itself (e.g., as a ribozyme or antisense sequence).

Retroviral vector constructs of the present invention may be readilyconstructed from a wide variety of retroviruses, including for example,B, C, and D type retroviruses as well as spumaviruses and lentiviruses(see, e.g., RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Such retroviruses may be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; Rockville, Md.), or isolated from known sources using commonlyavailable techniques. Any of the above retroviruses may be readilyutilized in order to assemble or construct retroviral vector constructs,packaging cells, or producer cells of the present invention given thedisclosure provided herein, and standard recombinant techniques (e.g.,Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Kunkle, 1985 PNAS 82:488).

Suitable promoters for use in viral vectors generally may include, butare not limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller, et al., 1989Biotechniques 7:980-990, or any other promoter (e.g., cellular promoterssuch as eukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters that maybe employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein, and may be from amongeither regulated promoters or promoters as described above.

As described above, the retroviral plasmid vector is employed totransduce packaging cell lines to form producer cell lines. Examples ofpackaging cells which may be transfected include, but are not limitedto, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE,ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller,Human Gene Therapy, 1:5-14 (1990). The vector may transduce thepackaging cells through any means known in the art. Such means include,but are not limited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

The producer cell line generates infectious retroviral vector particlesthat include the nucleic acid sequence(s) encoding the bindingdomain-immunoglobulin fusion polypeptides or fusion proteins. Suchretroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding the bindingdomain-immunoglobulin fusion polypeptide or fusion protein. Eukaryoticcells which may be transduced include, but are not limited to, embryonicstem cells, as well as hematopoietic stem cells, hepatocytes,fibroblasts, circulating peripheral blood mononuclear andpolymorphonuclear cells including myelomonocytic cells, lymphocytes,myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymphoidand reticuloendothelia cells of the lymph nodes and spleen,keratinocytes, endothelial cells, and bronchial epithelial cells.

As another example of an embodiment of the invention in which a viralvector is used to prepare, for example, a recombinant bindingdomain-immunoglobulin fusion expression construct, in one preferredembodiment, host cells transduced by a recombinant viral constructdirecting the expression of binding domain-immunoglobulin fusionpolypeptides or fusion proteins may produce viral particles containingexpressed binding domain-immunoglobulin fusion polypeptides or fusionproteins that are derived from portions of a host cell membraneincorporated by the viral particles during viral budding.

In another aspect, the present invention relates to host cellscontaining the herein described nucleic acid constructs, such as, forexample, recombinant binding domain-immunoglobulin fusion expressionconstructs. Host cells are genetically engineered (transduced,transformed or transfected) with the vectors and/or expressionconstructs of this invention that may be, for example, a cloning vector,a shuttle vector, or an expression construct. The vector or constructmay be, for example, in the form of a plasmid, a viral particle, aphage, etc. The engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants or amplifying particular genes such as genesencoding binding domain-immunoglobulin fusion polypeptides or bindingdomain-immunoglobulin fusion fusion proteins. The culture conditions forparticular host cells selected for expression, such as temperature, pHand the like, will be readily apparent to the ordinarily skilledartisan.

The host cell for production or expression of a construct of theinvention, for example, can be a higher eukaryotic cell, such as amammalian cell, or a lower eukaryotic cell, such as a yeast cell, or thehost cell can be a prokaryotic cell, such as a bacterial cell.Representative examples of appropriate host cells according to thepresent invention include, but need not be limited to, bacterial cells,such as E. coli, Streptomyces, Salmonella tvphimurium; fungal cells,such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9;animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells,or any suitable cell already adapted to in vitro propagation or soestablished de novo. The selection of an appropriate host is deemed tobe within the scope of those skilled in the art from the teachingsherein.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, 1981Cell 23:175, and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences, for example as described herein regarding the preparation ofbinding domain-immunoglobulin fusion expression constructs. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.Introduction of the construct into the host cell can be effected by avariety of methods with which those skilled in the art will be familiar,including but not limited to, for example, calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986 Basic Methods in Molecular Biology).

The present invention constructs, for example, bindingdomain-immunoglobulin fusion proteins, or compositions comprising one ormore polynucleotides encoding same as described herein (for example, tobe administered under conditions and for a time sufficient to permitexpression of a binding domain-immunoglobulin fusion protein in a hostcell in vivo or in vitro, for gene therapy, for example, among otherthings), may be formulated into pharmaceutical compositions foradministration according to well known methodologies. Pharmaceuticalcompositions generally comprise one or more recombinant expressionconstructs, and/or expression products of such constructs, incombination with a pharmaceutically acceptable carrier, excipient ordiluent. Such carriers will be nontoxic to recipients at the dosages andconcentrations employed. For nucleic acid-based formulations, or forformulations comprising expression products of the subject inventionrecombinant constructs, about 0.01 μg/kg to about 100 mg/kg body weightwill be adminstered, for example, typically by the intradermal,subcutaneous, intramuscular or intravenous route, or by other routes. Apreferred dosage, for example, is about 1 μg/kg to about 1 mg/kg, withabout 5 μg/kg to about 200 μg/kg particularly preferred. It will beevident to those skilled in the art that the number and frequency ofadministration will be dependent upon the response of the host.“Pharmaceutically acceptable carriers” for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compounds of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions that contain one or more nucleic acidconstructs of the invention, for example, binding domain-immunoglobulinfusion protein encoding constructs (or their expressed products) may bein any form that allows for the composition to be administered to apatient. For example, the composition may be in the form of a solid,liquid or gas (aerosol). Typical routes of administration include,without limitation, oral, topical, parenteral (e.g., sublingually orbuccally), sublingual, rectal, vaginal, and intranasal. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal, intracavernous, intrathecal, intrameatal,intraurethral injection or infusion techniques. The pharmaceuticalcomposition is formulated so as to allow the active ingredientscontained therein to be bioavailable upon administration of thecomposition to a patient. Compositions that will be administered to apatient take the form of one or more dosage units, where for example, atablet may be a single dosage unit, and a container of one or morecompounds of the invention in aerosol form may hold a plurality ofdosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to one or more binding domain-immunoglobulin fusion constructor expressed product, one or more of a sweetening agent, preservatives,dye/colorant and flavor enhancer. In a composition to be administered byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent, for example, may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

It may also be desirable to include other components in the preparation,such as delivery vehicles including but not limited to aluminum salts,water-in-oil emulsions, biodegradable oil vehicles, oil-in-wateremulsions, biodegradable microcapsules, and liposomes. Examples ofimmunostimulatory substances (adjuvants) for use in such vehiclesinclude N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon andIL-15.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic galactide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable that the microsphere be larger than approximately 25microns.

Pharmaceutical compositions may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

As described above, the subject invention includes compositions capableof delivering nucleic acid molecules encoding bindingdomain-immunoglobulin fusion proteins. Such compositions includerecombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (seeBerkner, 1988 Biotechniques 6:616-627; L1 et al., 1993 Hum. Gene Ther.4:403-409; Vincent et al., Nat. Genet. 5:130-134; and Kolls et al., 1994Proc. Natl. Acad. Sci. USA 91:215-219), pox virus (see U.S. Pat. No.4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)), recombinantexpression construct nucleic acid molecules complexed to a polycationicmolecule (see WO 93/03709), and nucleic acids associated with liposomes(see Wang et al., 1987 Proc. Natl. Acad. Sci. USA 84:7851). In certainembodiments, the DNA may be linked to killed or inactivated adenovirus(see Curiel et al., 1992 Hum. Gene Ther. 3:147-154; Cotton et al., 1992Proc. Natl. Acad. Sci. USA 89:6094). Other suitable compositions includeDNA-ligand (see Wu et al., 1989 J. Biol. Chem. 264:16985-16987) andlipid-DNA combinations (see Felgner et al., 1989 Proc. Natl. Acad. Sci.USA 84:7413-7417).

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from a host, modified, and placed into thesame or another host animal. It will be evident that one can utilize anyof the compositions noted above for introduction of constructs of theinvention, for example, binding domain-immunoglobulin fusion proteins orof binding domain-immunoglobulin fusion protein encoding nucleic acidmolecules into tissue cells in an ex vivo context. Protocols for viral,physical and chemical methods of uptake are well known in the art.

Accordingly, the present invention is useful for treating a patienthaving a B cell disorder or a malignant condition, or for treating acell culture derived from such a patient. As used herein, the term“patient” refers to any warm-blooded animal, preferably a human. Apatient may be afflicted with cancer or a malignant condition, such as Bcell lymphoma, or may be normal (i.e., free of detectable disease andinfection). A “cell culture” includes any preparation amenable to exvivo treatment, for example a preparation containing immunocompetentcells or isolated cells of the immune system (including, but not limitedto, T cells, macrophages, monocytes, B cells and dendritic cells). Suchcells may be isolated by any of a variety of techniques well known tothose of ordinary skill in the art (e.g., Ficoll-hypaque densitycentrifugation). The cells may (but need not) have been isolated from apatient afflicted with a B cell disorder or a malignant condition, andmay be reintroduced into a patient after treatment.

A liquid composition intended for either parenteral or oraladministration should contain an amount of a construct of the invention,for example, a binding domain-immunoglobulin fusion protein encodingconstruct or expressed product, such that a suitable dosage will beobtained. Typically, this amount is at least 0.01 wt % of a bindingdomain-immunoglobulin fusion construct or expressed product in thecomposition. When intended for oral administration, this amount may bevaried to be between 0.1 and about 70% of the weight of the composition.Preferred oral compositions contain between about 4% and about 50% ofbinding domain-immunoglobulin fusion construct or expressed product(s).Preferred compositions and preparations are prepared so that, forexample, a parenteral dosage unit contains between 0.01 to 1% by weightof active compound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment, or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of a construct of the invention, for example, a bindingdomain-immunoglobulin fusion construct or expressed product, of fromabout 0.1 to about 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository that will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

In the methods of the invention, a construct of the invention, forexample, a binding domain-immunoglobulin fusion encoding constructs orexpressed product(s), may be administered through use of insert(s),bead(s), timed-release formulation(s), patch(es) or fast-releaseformulation(s).

Constructs of the invention, for example, antigen-binding constructs ofthe invention, may be administered or co-administered to an animal orpatient in combination with, or at the same or about the same time, asother compounds. In one aspect, one or more constructs, including forexample one or more antigen-binding constructs, are administered to ananimal or patient in conjunction with one or more chemotheraputiccompounds such as alkylating agents, nucleoside analogues, and the like.The administration or co-administration of one or more constructs,including one or more antigen-binding constructs, of the invention andone or more chemotheraputic agents can be used for the treatment oftumors or cancer in an animal or patient. Exemplary cancers include, butare not limited to, head and neck cancer, breast cancer, colorectalcancer, gastric cancer, hepatic cancer, bladder cancer, cervical cancer,endometrial cancer, lung cancer (non-small cell), ovarian cancer,pancreatic cancer, prostate cancer; choriocarcinoma (lung cancer); hairycell leukemia, chronic lymphotic leukemia, acute lymphocytic leukemia(breast & bladder), acute myelogenous leukemia, meningeal leukemia,chronic myelogenous leukemia, erythroleukemia. More commonly the cancerstreated include non-Hodgkin's lymphoma (osteogenic sarcoma, adult softtissue sarcoma), T-cell lymphoma, chronic lymphocytic leukaemia, slowlygrowing non-Hodgkin's lymphomas, Hodgkin's lymphoma and ovarian cancer.

Examples of an alkylating agents that can be co-administered with one ormore constructs, including one or more antigen-binding constructs, ofthe invention include mechlorethamine, chlorambucil, ifosfamide,melphalan, busulfan, carmustine, lomustine, procarbazine, dacardazine,cisplatin, carboplatin, mitomycin C, cyclophosphamide, ifosfamide,thiotepa, and dacarbazine, and analogues thereof. See for example U.S.Pat. No. 3,046,301 describing the synthesis of chlorambucil, U.S. Pat.No. 3,732,340 describing the synthesis of ifosfamide, U.S. Pat. No.3,018,302 for the synthesis of cyclophosphamide, U.S. Pat. No. 3,032,584describing the synthesis of melphalan, and Braunwald et al., “Harrison'sPrinciples of Internal Medicine,” 15th Ed., McGraw-Hill, New York, N.Y.,pp. 536-544 (2001) for clinical aspects of cyclophosphamide,chlorambucil, melphalan, ifosfamide, procarbazine, hexamethylmelamine,cisplatin, and carboplatin. Examples of nucleoside analogues, include,but are not limited to, fludarabine pentostatin, methotrexate,fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine,floxuridine, mercaptopurine, 6-thioguanine, cladribine, and analoguesthereof. One example is the combination of constructs, includingantigen-binding constructs that bind CD20. This construct acts as achemosensitising agent and works together with chemotherapeutic agents,such that less chemotherapeutic agents are necessary to achieveanti-tumor or anti-cancer effects. For example, U.S. Pat. No. 3,923,785describing the synthesis of pentostatin, U.S. Pat. No. 4,080,325describing the synthesis of methotrexate, U.S. Pat. No. 2,802,005describing the synthesis of fluorouracil, and Braunwald et al.,“Harrison's Principles of Internal Medicine,” 15th Ed., McGraw-Hill, NewYork, N.Y., pp. 536-544 (2001) for clinical aspects of methotrexate,5-fluorouracil, cytosine arabinoside, 6-mercaptopurine, 6-thioguanine,and fludarabine phosphate.

In another aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-administered compounds that inhibit topoisomerase II or compoundsthat otherwise interact with nucleic acids in cells. Such compoundsinclude, for example, doxorubicin, epirubicin, etoposide, teniposide,mitoxantrone, and analogues thereof. In one example, this combination isused in treatment to reduce tumor cell contamination of peripheral bloodprogenitor cells (PBSC) in conjunction with high-dose chemotherapy andautologous stem cell support (HDC-ASCT). See U.S. Pat. No. 6,586,428 toGeroni et al.

In anther aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-adminstered with therapeutic drugs. For example, Virulizin (LorusTherapeutics), which is believed to stimulate the release of tumournecrosis factor, TNF-alpha, by tumour cells in vitro and stilumalateactiviation of macrophage cells. This can be used in combination withone or more constructs, including one or more antigen-bindingconstructs, of the invention to increase cancer cell apoptosis and treatvarious types of cancers including Pancreatic Cancer, MalignantMelanoma, Kaposi's Sarcoma (KS), Lung Cancer, Breast Cancer, Uterine,Ovarian and Cervical Cancer. Another example is CpG 7909 (ColeyPharmaceutical Group), which is believed to activate NK cells andmonocytes and enhance ADCC. This drug can be used in combination withcancer or tumor specific constructs, including antigen-bindingconstructs, of the invention, such as an anti-CD20 construct, to treatnon-Hodgkin's lymphoma and other cancers.

One or more constructs, including one or more antigen-bindingconstructs, of the invention can also be combined with angiogensisinhibitors to increase anti-tumor effects. Angiogenisis is the growth ofnew blood vessels. This process allows tumors to grow and metastasize.Inhibiting angiogeneisis can help prevent metastasis, and stop thespread of tumors cells. Angiogenisis inhibitors include, but are notlimited to, angiostatin, endostatin, thrombospondin, platelet factor 4,Cartilage-derived inhibitor (CDI), retinoids, Interleukin-12, tissueinhibitor of metalloproteinase 1, 2 and 3 (TIMP-1, TIMP-2, and TIMP-3)and proteins that block the angiogensis signaling cascade, such asanti-VEGF (Vascular Endothelial Growth Factor) and IFN-alpha.Angiogenesis inhibitors can be administered or co-administered withtumor specific constructs, including antigen-binding constructs capableof mediating, for example, ADCC and/or complement fixation orchemotherapy-conjugated antigen-binding of the invention to combatvarious types of cancers, for example, solid tumor cancers such as lungand breast cancer.

In another aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-administered with disease modifying anti-rheumatic agents (DMARagents) for the treatment of rheumatoid arthritis, psoriasis, ulcerativecolitus, systemic lupus erythematosus (SLE), Crohn's disease, ankylosingspondylitis, and various inflammatory disease processes. In suchtreatment, the constructs, for example, antigen-binding constructs, ofthe invention are commonly administered in conjunction with compoundssuch as azathioprine, cyclosporin, gold, hydroxychloroquine,methotrexate, penicallamine, sulphasalazine, and the like.

In another aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-administered with agents or compounds that counteract the biologicaleffects of interleukin-1, including for example interleukin-1 inhibitorsand interleukin-1 receptor antagonist. It is thought that interleukin-1has a role in the generation of rheumatoid arthritis (RA), inflammation,and the destruction of joints. IL-1 inhibitors can also be used inconjunction with the constructs, including antigen-binding constructs,of the invention to treat arthritis, inflammatory bowel disease, sepsisand septic shock, ischemic injury, reperfusion, ischemic brain injurysuch as cerebral palsy and multiple sclerosis. See U.S. Pat. No.6,159,460 to Thompson et al. In another aspect, for example, one or moreconstructs, including one or more antigen-binding constructs, of theinvention can be administered or co-administered to an animal or patientin conjunction with one or more glucocorticoids for example,methylprednisilone, dexamethasone, hydrocortisone, and the like.Glucocorticoids have been used to induce apoptosis and inhibit growth,independent of ADCC and CDC. These compounds can be combined withconstructs, including antigen-binding constructs, of the inventioncapable of inducing apoptosis in cancer cells. In one example is theanti-CD20, and anti-CD40 antigen-binding constructs, which can be usedto induce apoptosis in B-cells, are combined with glutcocorticoids totreat B-cell non-Hodgkin's lymphoma (NHL).

In another aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-administered with p38 inhibitors or antagonists. The p38mitogen-activated protein kinase pathway is involved in a number ofcellular processes instrumental to the development of rheumatoidarthritis. For example, the activation and infiltration of leukocytes aswell as the production of inflammatory cytokines are p38-dependentprocesses.

In another aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention are administered orco-administered with compounds that promote the differentiation andproliferation of B-cells. Cytokines such as \ interleukin-4 (IL-4) andinterleukin-6 (IL-6), in additional to other biological activities, havebeen shown to stimulate antibody synthesis and secretion by activated Blympocytes. In a particular aspect of the invention, constructs,including antigen-binding constructs that recognize and bind CD20 areco-administered with one or more of interleukin-4 (IL-4) andinterleukin-6 (IL-6).

In another aspect one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-administered with Interleukin-2 (IL-2). Interleukin 2 (IL-2) is alymphokine that increases production of effector cells, such as CD4+T-helper cells, CD8 cytotoxic cells, antibody producing B cells, naturalkiller cells (NK), and monocytes/macrophages. IL-2 helps produceT-cells, which in turn secrete more of the IL-2 (an “autocrine loop”).IL-2 can be used to augment antibody-dependent cell-mediatedcytotoxicity (ADCC) and immunotherapies associated with constructs ofthe invention. In one example, an anti-CD20 construct of the inventionand IL-2 are used to treat patients with relapsed or refractoryfollicular non-Hodgkin's lymphoma. In another example IL-2 isadministered or co-administered with HIV immunotherapies to help with Tcell recovery.

In another aspect one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-administered with Interleukin-12 (IL-12). IL-12 is know to enhancecytolytic T-cell responses, promote the development of helper T cells,enhance the activity of natural killer (NK) cells, and induces thesecretion of IFN-γ in T and NK cells. IL-12 also increases many helperand effector cells that mediate apoptosis. In another aspect of theinvention, one or more constructs, including one or more antigen-bindingconstructs, are administered or co-administeredwith IL-12 in thetreatment of an animal or patient with a tumor or cancer. For example, aconstruct, including an antigen-binding construct, of the invention thatbinds CD20 combined with IL-2 for thetreatment of a patient with B-cellnon-Hodgkin's lymphoma (NHL).

One or more constructs, including one or more antigen-bindingconstructs, of the invention can also be combined with immunomodulatorsto boost the efficacy of the antigen-binding constructs of theinvention. Immunomodulators include, but are not limited to, ColonyStimulating Factors (CSF), Tumor necrosis Factors (TNF), and Interferons(IFN).

CSFs can include granulocyte-macrophage CSF (GM-CSF), granulocyte-CSF(G-CSF), and macrophage CSF (M-CSF). GM-CSF is thought to regulate thedevelopment of neutrophils, macrophages, monocytes and eosinophils.G-CSF has been shown to induce neutrophil production, and M-CSFproduction. M-CSF has been shown to stimulate macrophages and monocytes.The use of CSFs to treat neutropenia in cancer patients has been longestablished. In one example, constructs, including antigen-bindingconstructs, of the invention can be combined with GM-CSF, G-CSF orcombinations thereof in order to accelerate recovery from neutropenia inpatients after bone marrow trans-plantation and chemotherapy.Neutrophils play a major role in fighting microbes such as bacterial,fungi and parasites. Patients with neutropenia are particularlysusceptible to bacterial and wide spread fungal infections. In anotherexample, a construct, including an antigen binding construct, of theinvention can be combined with GM-CSF-treated neutrophils, monocytes andmacrophages to increase activity against bacteria, fungi, etc, includingthe dreaded Pneumocystis carinii.

An example of an IFN is interferon alpha (IFN-α). IFN-α is madenaturally by some types of white blood cell as part of the immuneresponse when the body reacts to cancers or viral infections. It has twomain modes of attack, interfering with growth and proliferation ofcancer cells and it boosting the production of killer T cells and othercells that attack cancer cells. Interferon is also thought to facilitatecancer cells to put out chemical signals that make them better targetsfor the immune system, and has been used in recent years for severaldifferent types of cancer, particularly kidney cancer, melanoma,multiple myeloma, and some types of leukemia. It is also used to treatviral infections such as hepatitis. Interferon-alpha2a, for example,enhances ADCC and can be combined with one or more constructs, includingantigen-binding constructs, of the invention to increase the efficiencyof ADCC activity associated with the construct. In another example, oneor more constructs, including one or more antigen-binding constructs ofthe invention are administered or co-administered to an animal orpatient with interferon-gamma (IFN-γ), which has been show to increasethe number of anti-CD20 antigens on B cells and bone marrow plasma cells(BMPC). This is particularly useful for the treatment of patients withmultiple myelomas, which have a reduced expression of CD20 in their Bcells and bone marrow plasma cells (BMPC). Accordingly, the treatment ofmultiple myeloma patients with constructs, including antigen-bindingconstructs of the invention, in particular constructs that bind CD20,may be usefully co-administered in conjunction with IFN-γ TNF is a classof natural chemicals with anticancer properties. One example of a TNF isTNF-alpha. TNF-alpha has also been shown to have synergistic effectswith IFN-gamma and IL-12. In another example, TNF can be administered orco-administered with one or more tumor specific constructs, includingone or more antigen-binding constructs, of the invention, and includechemotherapy-conjugated antigen binding constructs of the invention,together with IFN-gamma, IL-12 or various combinations thereof. TNF isalso known to be an inflammatory regulation molecule. TNF-alphaantibodies or antagonist(s) can be combined with anti-T cell constructs,including antigen-binding constructs, of the invention to treat patientswith rheumatoid arthritis, psoriasis, ulcerative colitus, systemic lupuserythematosus (SLE), Crohn's disease, ankylosing spondylitis, andvarious inflammatory disease processes.

In another aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention can be administered orco-administered with another antibody or antigen-binding construct ofthe invention. One example is a construct, for example, anantigen-binding construct of the invention capable of binding CD20combined with a construct capable of binding CD22, CD19 or combinationsthereof. This combination is effective as a treatment for indolent andaggressive forms of B-cell lymphomas, and acute and chronic forms oflymphatic leukemias. See U.S. Pat. No. 6,306,393 to Goldberg. In anotherexample, constructs, including antigen-binding constructs, of theinvention are co-administered with other constructs such asantigen-binding constructs of the invention that aid in mediatingapoptosis. For example, a combination of one or more constructs,including one or more antigen-binding constructs of the inventioncapable of binding CD28, CD3, CD20 or a combination thereof. Thecombination of anti-CD28 and CD3 provides a method for prolongedproliferation of T-cells. See U.S. Pat. No. 6,352,694 to June et al.This prolonged T-cell proliferation increases the efficiency immunedependent cytotoxicity, particularly those associated with anti-CD20.

In another aspect, constructs, including antigen-binding constructs, ofthe invention can be administered or co-administered with one or moreT-cell regulatory molecules. One example is a combination withinterleukin-12 (IL-12). The IL-12 cytokine stimulates cell-mediatedimmunity, has angiostatic activity, and possesses significant anti-tumoreffects in a variety of tumor models. IL-12 has also been shown tostimulate the production of interferon-gamma (IFN-γ). Accordingly, thetreatment of multiple myeloma patients with one or more constructs,including one or more antigen-binding constructs, of the invention, inparticular those that bind CD20, is expected to be more efficacious whenco-administered in conjunction with IL-12. In another example, one ormore constructs, including one or more antigen-binding constructs, ofthe invention can be administered or co-administered with abinding-domain construct of the invention other protein capable ofbinding CTLA-4 to enhance the anti-tumor immune response, by inhibitingthe downregulation of T-cell activation.

In another aspect, one or more constructs, including one or moreantigen-binding constructs, of the invention can be combined with genetherapies. In one example, a chemotherapy-conjugated construct of theinvention is administered or co-administered with the Bcl-2 antisenseoligonucleotide. Bcl-2 is associated with tumor resistance toanti-cancer therapies, and its believed to blocking chemotherapy-inducedcell death. In another example one or more constructs, including one ormore antigen-binding constructs, of the invention is administered orco-administered with an adenovirus for delivery of a “suicide gene.” Theadenovirus inserts the gene directly into the tumor cells, which makesthese cells sensitive to an otherwise ineffective drug. Drug treatmentthen destroys the tumor cells, while leaving healthy cells untouched.However, once therapy is complete stray cancer cells that escapedtherapy can reestablish and metastasize. Combining gene therapy with oneor more constructs, including one or more antigen-binding constructs,will help kill stray cancer cells and minimize cancer reoccurrence.

A similar combination can be used with palliative (non-radical)operations to surgically remove tumors. In this example one or moreconstructs, including one or more antigen-binding constructs, of theinvention can be administered before and after surgical extractions oftumors in order to increase the immune response and reduce thelikelihood of reoccurrence by killing any cancer cells that were notremoved during the surgery.

Another aspect combines a cancer or antigen vaccine and T-cell regulatormolecules. For example, the binding portion, for example, anantigen-binding portion, of a construct can be specific for a cancercell or antigen, or a protein fragment from a cancer cell or antigen.This can help mediate an immune response against a particular tumor orantigen. Such constructs can be combined with T-cell regulators toincrease the efficiency of the immune response.

In another example, one or more constructs, including one or moreantigen-binding constructs, of the invention is administered orco-administered with retinoids. Retinoids include Vitamin A and itsderivatives, which have the ability to stop cells from dividing andcause them to differentiate. Vitamin A is combined with an anti-cancerconstruct(s), including antigen-binding construct(s), of the inventionto combat various forms of cancer.

The terms “binding construct” and “antigen-binding construct” as usedherein may refer to, for example, engineered polypeptides, recombinantpolypeptides, synthetic, semi-synthetic or other fusion proteins thatare capable of binding a target, for example, an antigen.Antigen-binding constructs of the invention may be used in variousapplications, including those within the variety of uses to whichantibodies or related immunoglobulin-type constructs may be put.Constructs, including antigen-binding constructs of the invention can beused in in vivo and in vitro experiments for therapeutic, diagnostic,research, and other purposes. Such uses include, for example, thefollowing.

Constructs, including antigen-binding constructs of the invention may beused for immunohistochemistry applications. For example, they may beused for immunolocalization of a particular antigen or group of antigensin a tissue. Tissue can be fixed and incubated with antigen-bindingconstructs of interest. These constructs can then be localized using asecondary antibody or binding construct of the invention coupled to alabel, for example, to a gold particle or an enzyme that gives achemical reaction, like horseradish peroxidase or beta-galactosidase. Asecondary antibody or binding construct is frequently made that isreactive against, for example, a portion of the primary bindingconstruct. Thus, for example, if the primary binding construct has ahuman tail portion, the secondary antibody or binding construct couldbe, for example, a rabbit anti-mouse antibody or antigen-bindingconstruct that has been linked to beta-galactosidase. Alternatively theantibody or binding construct of the invention can be purified and thenconjugated to another molecule to produce a fluorescent antibody orbinding construct.

Constructs, including antigen-binding contracts of the invention canalso be used to detect the location of an antigen or antigens on thesurface of cells or to detect the location of intracellular materialsusing, for example, Immunoelectron Microscopy. Electron dense materialssuch as ferritin or colloidal gold, for example, can be conjugated to anantigen-binding construct. Scanning electron microscopy can be used todetect the localization of the antigen/binding construct complex.

Constructs, including antigen-binding constructs of the invention mayalso be used to quantitate the presence of an antigen or antigens usingone of a variety of immunoassay formats, for example, a radioimmunoassay(RIA) format or an enzyme-linked immunosorbent assay (ELISA) format.There are many variants of these approaches, but those are based on asimilar idea. For example, if an antigen can be bound to a solid supportor surface, or is in solution, it can be detected by reacting it with aspecific antigen-binding construct of the invention. The presence oramount of the construct can then be detected or quantitated by reactingit with, for example, either a secondary antibody or a secondantigen-binding construct of the invention by incorporating a labeldirectly into the primary antibody. Alternatively, for example, anantigen-binding polypeptide of the invention can be bound to a solidsurface and the antigen added. A second antibody or antigen-bindingpolypeptide(s) of the invention that recognizes a distinct epitope onthe antigen can then be added and detected. This technique is commonlyreferred to as a “sandwich assay”, which is frequently used to avoidproblems of high background or non-specific reactions, among otherreasons.

Because the binding constructs of the invention can have highaffinity/affinities and/or selectivity/selectivities for a particularepitope or epitopes, they can also be used as affinity reagents, forexample, in protein or antigen purification. In one example of such aprocess, antigen-binding constructs of the invention are immobilized ona suitable support, for example, Sephadex resin or filter paper. Theimmobilized construct is exposed to a sample containing, or suspected ofcontaining, a target protein(s) or antigen(s). The support is rinsedwith a suitable buffer that will remove unwanted materials. The supportis washed with another buffer that will release the bound protein(s) orantigen(s).

Because particular binding constructs of the invention can bind toproteins or other antigens with high affinity and selectivity they canalso be used as a criterion for the importance of a particular enzyme orother macromolecule in a particular reaction. If an antigen-bindingconstruct of the invention can interfere with a reaction in a solution,this will indicate that the construct may be binding specifically to aprotein or other antigenic material involved in that reaction.

Constructs, including antigen-binding constructs of the invention canalso be used as receptor blockers or inhibitors or antagonists.

Constructs, including antigen-binding constructs of the invention canalso be used in identifying and studying the function(s) of proteins. Ifan antigen-binding construct of the invention reacts with a specificprotein, for example, that protein can subsequently be precipitated fromsolution, for example. Precipitation is typically performed by using asecondary antibody or antigen-binding construct of the invention thatlinks primary complexes together. Alternatively, the complex can beremoved by reacting the solution with either protein A or, for example,depending on the construct, an anti-Fc antibody, for example, which hasbeen attached to beads, for example, so that can be easily removed formthe solution.

Constructs, including antigen-binding constructs of the invention canalso be used in conjunction with gel-shift experiments to identifyspecific nucleic acid-binding proteins such as DNA-binding proteins. Forexample, DNA-binding proteins can be assayed by their ability to bindwith high affinity to a particular oligonucleotide. The mobility of anoligonucleotide associated with the protein is far different than themobility of a free oligonucleotide and results in a gel migrationpattern and signal that is commonly referred to as a gel shift. Theaddition of the construct to the binding assay can have either of twoeffects. If the construct binds to a region of a protein not involved inDNA binding it can result in a complex that has even a slower mobilityand is detected as a greater shift in mobility (a super-shift).Alternatively, if the construct binds to a region of the proteininvolved in recognizing the DNA then it can disrupt the binding andeliminate the shift. In either case, the data from these experiments canserve as a criterion to identify a DNA-binding protein, for example.

It is also possible to use constructs, including antigen-bindingconstructs of the invention to detect a protein by western blottingafter fractionation by SDS-PAGE, for example. Once fractionated proteinsare transferred to a membrane such as a nitrocellulose sheet, they areexposed to a particular antigen-binding construct of the invention thatspecifically recognizes, or recognizes to a desired degree ofselectivity, proteins immobilized to the blot. This allows particularproteins to be identified. This approach is particularly useful if themobility of the protein changes during an experiment. For example,incorporation of a phosphate or a carbohydrate, or cleavage of theprotein, results in a change in mobility that can be followed instraightforward manner by western analysis. With appropriate controls,this approach can be used to measure the abundance of a protein inresponse to experimental manipulations.

The combination of SDS gels and immunoprecipitation can also beextremely effective. If a particular protein can be immunoprecipitatedin a solution, both supernatant and precipitated fractions can beseparated on an SDS gel and studied using an antigen-bindingconstruct(s) of the invention.

Sometimes a binding construct of the invention directed against oneprotein will also precipitate a second protein that interacts with thefirst protein. The second protein, as well as the first, can then beseen by staining the gel or by autoradiography. This relationship isfrequently the first indication that a protein functions as part of acomplex and it can also be used to demonstrate a physical interaction oftwo proteins that are hypothesized to interact on the basis of otherevidence (e.g., a two hybrid screen or a supressor mutation). Thisapproach can be combined with western blotting analysis in severalextremely effective ways.

Thus, for example, antigen-binding constructs of the invention can beused in a combination of immunoprecipitation and western analysis in thestudy, for example, of signal transduction and protein processing. Forexample, an immunoprecipitated protein can be subsequently studied bywestern analysis using a different antibody or antigen-binding constructof the invention that binds to the protein. The most useful of are thosethat are directed against particular structural determinants that may bepresent in a protein. Thus, an antibody or antigen-binding construct ofthe invention directed against a region of the protein that undergoesproteolytic processing can be useful to follow proteolytic processing.Additionally, a construct of the invention or a mixture ofantigen-binding constructs of the invention that recognizephosphorylated peptides (e.g., anti PY (phosphorylated tyrosine) can beused to follow the extent of phosphorylation of a protein (using westernanalysis) after it is precipitated, or visa versa. Glycosylationreactions can also be followed by antigen-binding constructs of theinvention directed against a carbohydrate epitope (or by lectins, i.e.,proteins that recognize carbohydrates). Likewise, some antigen-bindingconstructs of the invention can be made that specifically recognize aphosphorylated epitope, for example, that will recognize a tyrosine or aserine residue after phosphorylation, but will not bind (or detectablybind) the epitope in the absence of phosphate. This approach can be usedto determine the phosphorylation state of a particular protein. Forexample, the phosphorylation of CREB (the cAMP response element bindingprotein) can be followed by an antibody that specifically recognizes anepitope in a way that is dependent on the phosphorylation of serine 133.

Constructs, including antigen-binding constructs of the invention canalso be used to screen expression libraries to isolate candidatepolynucleotides that express or present a particular epitope, or thathave a particular affinity or expression characteristic.

Constructs, including antigen-binding constructs of the invention thatbind to a cell surface can also be used as a marker to quantitate thefraction of cells expressing that marker using flow cytometry. Ifdifferent antigen binding constructs of the invention/fluorescent dyecombinations are used, for example, the fraction of cells expressingseveral antigens can be determined.

Constructs, including antigen-binding constructs of the invention thatfunction like anti-idiotype antibodies, i.e., antibodies against thebinding domain of another antibody, can be used in any of a number ofmethods in which is would be desirable or useful to mimic the structureof an antigen. Such uses include, for example, uses as cancer vaccines(including antigen-binding constructs of the invention that incorporatea molecular adjuvant), as probes for receptors, as receptor agonists, asreceptor antagonists, as receptor blockers or inhibitors, and so on.

In another aspect, constructs, including antigen-binding constructs ofthe invention may bispecific and thus capable of binding to two distinctepitopes, which may be present on the same or different cell types.

In vivo uses of constructs of the invention, including antigen-bindingconstructs, include therapy, alone or in combination with one or moreother therapies, for various diseases including cancers as well asB-cell disorders including autoimmune diseases. In some cases theconstructs of the invention are administered to a patient. In othercases, the construct may be coupled to another molecule by techniquesknown in the art, for example, a fluorescent molecule to aid in imaginga target, or a therapeutic drug and/or a toxin to aid in killing atarget.

For example, a labeling molecule or atom can be conjugated or otherwiselinked to the antigen-binding construct of the invention to aid inimaging or as a diagnostic agent. These include, but are not limited toenzymatic labels, radioisotopes or radioactive compounds or elements,fluorescent compounds or metals, chemiluminescent compounds andbioluminescent compounds. Thus, binding contructs or antigen-bindingconstructs of the invention can be conjugated to a drug, which allowsspecific drug targeting and increased efficiency once the drug reachesthe target. This facilitates drug therapy while reducing systemictoxicity and side effects. This allows use of drugs that would otherwisebe unacceptable when administered systemically. Dosage will depend onthe potency of the drug and the efficiency of the carrier construct.Other examples of in vivo uses include the use of binding constructs orantigen-binding constructs of the invention in which a toxin ischemically linked or conjugated to an polypeptide of the invention toform, for example, molecules that may be termed “immunoconjugates” or“immunotoxins.” Typically, for example, such a toxin may include one ormore radioisotopes (for example, Iodine-131, Yttrium-90, Rhenium-186,Copper-67, and/or Bishmuth-212), natural toxins, chemotherapy agents,biological response modifiers, or any other substance that is capable ofassisting in damaging or killing a target cell, inhibiting target cellreplication, or is effective in disrupting a desired cellular functionin a target cell.

The toxin portion of the immunotoxin can be derived form varioussources. Toxins are commonly derived from plants or bacteria, but toxinsof human origin or synthetic toxins can be used as well, for example.Examples of toxins derived from bacteria or plants include, but are notlimited to, abrin, α-sarcin, diptheria toxin, ricin, saporin, andpseudomonas exotoxin. Examples of mammalian enzymes include, but are notlimited to, ribonucleases (RNAse) and deoxyribonucleases. Numerousimmunotoxins that may be used with one or more constructs of theinvention have been described in the art. See, for example, U.S. Pat.No. 4,753,894 to Frankel et al.; U.S. Pat. No. 6,099,842 to Pastan etal.; Nevelle, et al., 1982 Immunol Rev. 62:75-91; Pastan et al., 1992Ann Rev Biochem 61:331-354; Chaudary et al., 1989 Nature 339:394; andBatra et al., 1991 Mol. Cell. Biol. 11:2200. Modified toxins describedherein and those described in the various publications are also withinthe scope of the instant invention.

Generally, the immunotoxins and other therapeutic agents of thisinvention are administered at a concentration that is therapeuticallyeffective to treat or prevent a particular disease, disorder, orcondition, such as for the treatment of tumors and malignancies, thetreatment of autoimmune diseases, allergies and inflammation, etc. Thiseffective dosage and mode of administration will depend on the animal orpatient being treated, the disease or condition being treated, thestrength of the immunoconjugates or immunotoxins and the efficiency ofthe conjugate. To accomplish this goal, the immunotoxins may beformulated using a variety of acceptable formulations and excipientsknown in the art. Typically, for example, the immunotoxins areadministered by injection, either intravenously or intraperitoneally.Methods to accomplish this administration are known to those of ordinaryskill in the art. It another aspect, the invention includes topically ororally administered compositions such as an aerosol or cream or patchthat may be capable of transmission across mucous membranes.

Formulants may be added to an immunoconjugates or immunotoxins of theinvention before administration to a patients being treated. A liquidformulation is most common, but other formulations are within the scopeof the invention. The formulants may include for example oils, polymers,vitamins, carbohydrates, amino acids, salts, buffers, albumin,surfactants, or bulking agents. Carbohydrates can include sugar or sugaralcohols such as mono, di, or polysaccharides, or water-soluble glucans.The saccharides or glucans can include for example fructose, dextrose,lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran,pullulan, dextrin, alpha and beta cyclodextrin, soluble starch,hydroxethyl starch and carboxymethylcellulose, or mixtures thereof“Sugar alcohol” may be defined as a C₄ to C₈ hydrocarbon having an —OHgroup and includes, for example, galactitol, inositol, mannitol,xylitol, sorbitol, glycerol, and arabitol. These sugars or sugaralcohols mentioned above may be used individually or in combination.There is no fixed limit to the amount used as long as the sugar or sugaralcohol is soluble in the aqueous preparation. In one aspect, the sugaror sugar alcohol concentration is between 0.5 w/v % and 15 w/v %,typically between 1.0 w/v % and 7.0 w/v %, more typically between 2.0and 6.0 w/v %.

Exemplary amino acids include levorotary (L) forms of camitine,arginine, and betaine; however, other amino acids may be added. Commonlyused polymers include polyvinylpyrrolidone (PVP) with an averagemolecular weight between 2,000 and 3,000, for example, or polyethyleneglycol (PEG) with an average molecular weight between 3,000 and 5,000,for example. A buffer can be used in the composition to minimize pHchanges in the solution before lyophilization or after reconstitution.Any physiological buffer may be used, but citrate, phosphate, succinate,and glutamate buffers or mixtures thereof are more commonly utilized.The concentration can be, for example, from 0.01 to 0.3 molar. Higher orlower concentrations may be used.

Immunotoxins of the invention can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Exemplary polymers and methods to attach them to peptides arereferenced in U.S. Pat. Nos. 4,766,106 to Katre et al.; 4,179,337 toDavis et al.; 4,495,285 to Shimizu et al.; and 4,609,546 to Hiratani.

The following Examples are offered by way of illustration and not by wayof limitation.

Example 1 Cloning of the 2H7 Variable Regions and Construction andSequencing of 2H7scFv-Ig

This Example illustrates the cloning of cDNA molecules that encode theheavy chain and light chain variable regions of the monoclonal antibody2H7. This Example also demonstrates the construction, sequencing, andexpression of 2H7scFv-Ig.

Prior to harvesting, cells expressing 2H7 monoclonal antibody thatspecifically bound to CD20 were kept in log phase growth for severaldays in RPMI 1640 media Invitrogen/Life Technologies, Gaithersburg, Md.)supplemented with glutamine, pyruvate, DMEM non-essential amino acids,and penicillin-streptomycin. Cells were pelleted by centrifugation fromthe culture medium, and 2×10⁷ cells were used to prepare RNA. RNA wasisolated from the 2H7-producing hybridoma cells using the Pharmingen(San Diego, Calif.) total RNA isolation kit (Catalog #45520K) accordingto the manufacturer's instructions accompanying the kit. One microgram(1 μg) of total RNA was used as template to prepare cDNA by reversetranscription. The RNA and 300 ng random primers were combined anddenatured at 72° C. for 10 minutes prior to addition of enzyme.Superscript II reverse transcriptase (Life Technologies) was added tothe RNA plus primer mixture in a total volume of 25 μl in the presenceof 5× second strand buffer and 0.1 M DTT provided with the enzyme. Thereverse transcription reaction was allowed to proceed at 42° C. for onehour.

The 2H7 cDNA generated in the randomly primed reverse transcriptasereaction and V region specific primers were used to amplify by PCR thevariable regions for the light and heavy chain of the 2H7 antibody. TheV region specific primers were designed using the published sequence(Genbank accession numbers M17954 for V_(L) and M17953 for V_(H)) as aguide. The two variable chains were designed with compatible endsequences so that an scFv could be assembled by ligation of the two Vregions after amplification and restriction enzyme digestion.

A (Gly₄Ser)₃ peptide linker to be inserted between the two V regions wasincorporated by adding the extra nucleotides to the antisense primer forthe V_(L) of 2H7. A Sac I restriction site was also introduced at thejunction between the two V regions. The sense primer used to amplify the2H7 V_(L), that included a HindIII restriction site and the light chainleader peptide was 5′-gtc aag ctt gcc gcc atg gat ttt caa gtg cag attttt cag c-3′ (SEQ ID NO:530). The antisense primer was 5′-gtc gtc gagctc cca cct cct cca gat cca cca ccg ccc gag cca ccg cca cct ttc agc tccagc ttg gtc cc-3′ (SEQ ID NO:531). The reading frame of the V region isindicated as a bold, underlined codon. The Hind III and Sad sites areindicated by underlined italicized sequences.

The V_(H) domain was amplified without a leader peptide, but included a5′ Sad restriction site for fusion to the V_(L) and a BclI restrictionsite at the 3′ end for fusion to various tails, including the human IgG1Fc domain and the truncated forms of CD40 ligand, CD154. The senseprimer was 5′-gct get gag ctc tca ggc tta tct aca gca agt ctg g-3′ (SEQID NO:532). The SacI site is indicated in italicized and underlinedfont, and the reading frame of the codon for the first amino acid of theV_(H) domain is indicated in bold, underlined type. The antisense primerwas 5′-gtt gtc tga tca gag acg gtg acc gtg gtc cc-3′ (SEQ ID NO:533).The BclI site is indicated in italicized, underlined type, and the lastserine of the V_(H) domain sequence is indicated in bold, underlinedtype.

The scFv-Ig was assembled by inserting the 2H7 scFv HindIII-BclIfragment into pUC19 containing the human IgG1 hinge, CH2, and CH3regions, which was digested with restriction enzymes, HindIII and BclI.After ligation, the ligation products were transformed into DH5abacteria. Positive clones were screened for the properly insertedfragments using the Sad site at the V_(L)-V_(H) junction of 2H7 as adiagnostic site. The 2H7scFv-Ig cDNA was subjected to cycle sequencingon a PE 9700 thermocycler using a 25-cycle program by denaturing at 96°C. for 10 seconds, annealing at 50° C. for 30 seconds, and extending at72° C. for 4 minutes. The sequencing primers were pUC forward andreverse primers and an internal primer that annealed to the CH2 domainhuman in the IgG constant region portion. Sequencing reactions wereperformed using the Big Dye Terminator Ready Sequencing Mix (PE-AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. Samples were subsequently purified using Centrisep columns(Catalog #CS-901, Princeton Separations, Adelphia, N.J.), the eluatesdried in a Savant vacuum dryer, denatured in Template SuppressionReagent (PE-ABI), and analyzed on an ABI 310 Genetic Analyzer(PE-Applied Biosystems). The sequence was edited, translated, andanalyzed using Vector Nti version 6.0 (Informax, North Bethesda, Md.).FIG. 1 shows the cDNA and predicted amino acid sequence of the2H7scFv-Ig construct.

Example 2 Expression of 2H7 ScFv-Ig in Stable CHO Cell Lines

This Example illustrates expression of 2H7scFv-Ig in a eukaryotic cellline and characterization of the expressed 2H7scFv-Ig by SDS-PAGE and byfunctional assays, including ADCC and complement fixation.

The 2H7scFv-Ig HindIII-XbaI (˜1.6 kb) fragment with correct sequence wasinserted into the mammalian expression vector pD18, and DNA frompositive clones was amplified using QIAGEN plasmid preparation kits(QIAGEN, Valencia, Calif.). The recombinant plasmid DNA (100 μg) wasthen linearized in a nonessential region by digestion with AscI,purified by phenol extraction, and resuspended in tissue culture media,Excell 302 (Catalog #14312-79P, JRH Biosciences, Lenexa, Kans.). Cellsfor transfection, CHO DG44 cells, were kept in logarithmic growth, and10⁷ cells harvested for each transfection reaction. Linearized DNA wasadded to the CHO cells in a total volume of 0.8 ml for electroporation.

Stable production of the 2H7 scFv-Ig fusion protein (SEQ. ID NO:10) wasachieved by electroporation of a selectable, amplifiable plasmid, pD18,containing the 2H7 scFv-Ig cDNA under the control of the CMV promoter,into Chinese Hamster Ovary (CHO) cells (all cell lines from AmericanType Culture Collection, Manassas, Va., unless otherwise noted). The 2H7expression cassette was subcloned downstream of the CMV promoter intothe vector multiple cloning site as a ˜1.6 kb HindIII-XbaI fragment. ThepD18 vector is a modified version of pcDNA3 encoding the DHFR selectablemarker with an attenuated promoter to increase selection pressure forthe plasmid. Plasmid DNA was prepared using Qiagen maxiprep kits, andpurified plasmid was linearized at a unique AscI site prior to phenolextraction and ethanol precipitation. Salmon sperm DNA (Sigma-Aldrich,St. Louis, Mo.) was added as carrier DNA, and 100 μg each of plasmid andcarrier DNA was used to transfect 10⁷ CHO DG44 cells by electroporation.Cells were grown to logarithmic phase in Excell 302 media (JRHBiosciences) containing glutamine (4 mM), pyruvate, recombinant insulin,penicillin-streptomycin, and 2×DMEM nonessential amino acids (all fromLife Technologies, Gaithersburg, Md.), hereafter referred to as “Excell302 complete” media. Media for untransfected cells also contained HT(diluted from a 100× solution of hypoxanthine and thymidine)(Invitrogen/Life Technologies). Media for transfections under selectioncontained varying levels of methotrexate (Sigma-Aldrich) as selectiveagent, ranging from 50 nM to 5 μM. Electroporations were performed at275 volts, 950 μF. Transfected cells were allowed to recover overnightin non-selective media prior to selective plating in 96 well flat bottomplates (Costar) at varying serial dilutions ranging from 125 cells/wellto 2000 cells/well. Culture media for cell cloning was Excell 302complete, containing 100 nM methotrexate. Once clonal outgrowth wassufficient, serial dilutions of culture supernatants from master wellswere screened for binding to CD20-CHO transfected cells. The clones withthe highest production of the fusion protein were expanded into T25 andthen T75 flasks to provide adequate numbers of cells for freezing andfor scaling up production of the 2H7scFvIg. Production levels werefurther increased in cultures from three clones by progressiveamplification in methotrexate containing culture media. At eachsuccessive passage of cells, the Excell 302 complete media contained anincreased concentration of methotrexate, such that only the cells thatamplified the DHFR plasmid could survive.

Supernatants were collected from CHO cells expressing the 2H7scFv-Ig,filtered through 0.2 μm PES express filters (Nalgene, Rochester, N.Y.)and were passed over a Protein A-agarose (IPA 300 crosslinked agarose)column (Repligen, Needham, Mass.). The column was washed with PBS, andthen bound protein was eluted using 0.1 M citrate buffer, pH 3.0.Fractions were collected and eluted protein was neutralized using 1MTris, pH 8.0, prior to dialysis overnight in PBS. Concentration of thepurified 2H7scFv-Ig was determined by absorption at 280 nm. Anextinction coefficient of 1.77 was determined using the protein analysistools in the Vector Nti Version 6.0 Software package (Informax, NorthBethesda, Md.). This program uses the amino acid composition data tocalculate extinction coefficients.

Production levels of 2H7scFv-Ig by transfected, stable CHO cells wereanalyzed by flow cytometry. Purified 2H7scFv-Ig to CHO cells was allowedto bind to CHO cells that expressed CD20 (CD20 CHO) and analyzed by flowcytometry using a fluorescein-conjugated anti-human IgG second stepreagent (Catalog Numbers H10101 and H10501, CalTag, Burlingame, Calif.).FIG. 2 (top) shows a standard curve generated by titration of 2H7scFv-Igbinding to CD20 CHO. At each concentration of 2H7scFv-Ig, the meanbrightness of the fluorescein signal in linear units is shown.Supernatants collected from T flasks containing stable CHO cell clonesexpressing 2H7scFv-Ig were then allowed to bind to CD20 CHO and thebinding was analyzed by flow cytometry. The fluorescein signal generatedby 2H7scFv-Ig contained in the supernatants was measured and the2H7scFv-Ig concentration in the supernatants was calculated from thestandard curve (FIG. 2, bottom).

Purified 2H7scFv-Ig was analyzed by electrophoresis onSDS-Polyacrylamide gels. Samples of 2H7scFv-Ig, purified by independentProtein A Agarose column runs, were boiled in SDS sample buffer withoutreduction of disulfide bonds and applied to SDS 10% Tris-BIS gels(Catalog #NP0301, Novex, Carlsbad, Calif.). Twenty micrograms of eachpurified batch was loaded on the gels. The proteins were visualizedafter electrophoresis by Coomassie Blue staining (Pierce Gel Code BlueStain Reagent, Catalog #24590, Pierce, Rockford, Ill.), and destainingin distilled water. Molecular weight markers were included on the samegel (Kaleidoscope Prestained Standards, Catalog #161-0324, Bio-Rad,Hercules, Calif.). The results are presented in FIG. 3. The numbersabove the lanes designate independent purification batches. Themolecular weights in kilodaltons of the size markers are indicated onthe left side of the figure. Further experiments with alternative samplepreparation conditions indicated that reduction of disulfide bonds byboiling the protein in SDS sample buffer containing DTT or2-mercaptoethanol caused the 2H7scFv-Ig to aggregate.

Any number of other immunological parameters may be monitored usingroutine assays that are well known in the art. These may include, forexample, antibody dependent cell-mediated cytotoxicity (ADCC) assays,secondary in vitro antibody responses, flow immunocytofluorimetricanalysis of various peripheral blood or lymphoid mononuclear cellsubpopulations using well established marker antigen systems,immunohistochemistry or other relevant assays. These and other assaysmay be found, for example, in Rose et al. (Eds.), Manual of ClinicalLaboratory Immunology, 5^(th) Ed., a 1997 American Society ofMicrobiology, Washington, D.C.

The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence ofcomplement was tested using B cell lines Ramos and Bjab. Rabbitcomplement (Pel-Freez, Rogers, Ak.) was used in the assay at a finaldilution of 1/10. Purified 2H7scFv-Ig was incubated with B cells andcomplement for 45 minutes at 37° C., followed by counting of live anddead cells by trypan blue exclusion. The results in FIG. 4A show that inthe presence of rabbit complement, 2H7scFv-Ig lysed B cells expressingCD20.

The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence ofperipheral blood mononuclear cells (PBMC) was tested by measuring therelease of ⁵¹Cr from labeled Bjab cells in a 4-hour assay using a 100:1ratio of PBMC to Bjab cells. The results shown in FIG. 4B indicated that2H7scFv-Ig can mediate antibody dependent cellular cytotoxicity (ADCC)because the release of ⁵¹Cr was higher in the presence of both PBMC and2H7scFv-Ig than in the presence of either PBMC or 2H7scFv-Ig alone.

Example 3 Effect of Simultaneous Ligation of CD20 and CD40 on Growth ofNormal B Cells, and on CD95 Expression, and Induction of Apoptosis

This Example illustrates the effect on cell proliferation ofcross-linking of CD20 and CD40 expressed on the cell surface.

Dense resting B cells were isolated from human tonsil by a Percoll stepgradient and T cells were removed by E-rosetting. Proliferation ofresting, dense tonsillar B cells was measured by uptake of³[H]-thymidine during the last 12 hours of a 4-day experiment.Proliferation was measured in quadruplicate cultures with means andstandard deviations as shown. Murine anti-human CD20 monoclonal antibody1F5 (anti-CD20) was used alone or was cross-linked with anti-murine κmonoclonal antibody 187.1 (anti-CD20XL). CD40 activation wasaccomplished using soluble human CD154 fused with murine CD8 (CD154)(Hollenbaugh et al., EMBO J. 11: 4212-21 (1992)), and CD40 cross-linkingwas accomplished using anti-murine CD8 monoclonal antibody 53-6(CD154XL). This procedure allowed simultaneous cross-linking of CD20 andCD40 on the cell surface. The results are presented in FIG. 5.

The effect of CD20 and CD40 cross-linking on Ramos cells, a B lymphomacell line, was examined. Ramos cells were analyzed for CD95 (Fas)expression and percent apoptosis eighteen hours after treatment (no goatanti-mouse IgG (GAM)) and/or cross-linking (+GAM) using murinemonoclonal antibodies that specifically bind CD20 (1F5) and CD40(G28-5). Control cells were treated with a non-binding isotype control(64.1) specific for CD3.

Treated Ramos cells were harvested, incubated with FITC-anti-CD95, andanalyzed by flow cytometry to determine the relative expression level ofFas on the cell surface after CD20 or CD40 cross-linking. Data isplotted as mean fluorescence of cells after treatment with the stimuliindicated (FIG. 6A).

Treated Ramos cells from the same experiment were harvested and bindingof annexin V was measured to indicate the percentage apoptosis in thetreated cultures. Apoptosis was measured by binding of Annexin V 18hours after cross-linking of CD20 and CD40 using 1F5 and G28-5 followedby cross-linking with GAM. Binding of Annexin V was measured using aFITC-Annexin V kit (Catalog #PN-IM2376, Immunotech, Marseille, France,).Annexin V binding is known to be an early event in progression of cellsinto apoptosis. Apoptosis, or programmed cell death, is a processcharacterized by a cascade of catabolic reactions leading to cell deathby suicide. In the early phase of apoptosis, before cells changemorphology and hydrolyze DNA, the integrity of the cell membrane ismaintained but cells lose the asymmetry of their membrane phospholipids,exposing negatively charged phospholipids, such as phosphatidylserine,at the cell surface. Annexin V, a calcium and phopholipid bindingprotein, binds preferentially and with high affinity tophosphatidylserine. Results demonstrating the effect of cross-linkingboth CD20 and CD40 on expression of the FAS receptor (CD95) arepresented in FIG. 6B. The effect of cross-linking of both CD20 and CD40on Annexin V binding to cells is shown in FIG. 6B.

Example 4 Construction and Characterization of 2H7 ScFv-CD154 FusionProteins

To construct a molecule capable of binding to both CD20 and CD40, cDNAencoding the 2H7 scFv was fused with cDNA encoding CD154, the CD40ligand. The 2H7 scFv cDNA encoded on the HindIII-BclI fragment wasremoved from the 2H7 scFvIg construct, and inserted into a pD18 vectoralong with a BamHI-XbaI cDNA fragment encoding the extracellular domainof human CD154. The extracellular domain is encoded at the carboxyterminus of CD154, similar to other type II membrane proteins.

The extracellular domain of human CD154 was PCR amplified using cDNAgenerated with random primers and RNA from human T lymphocytes activatedwith PHA (phytohemagglutinin). The primer sets included two different 5′or sense primers that created fusion junctions at two differentpositions within the extracellular domain of CD154. Two different fusionjunctions were designed that resulted in a short or truncated form (formS4) including amino acids 108 (Glu)-261 (Leu)+(Glu), and a long orcomplete form (form L2) including amino acids 48 (Arg)-261 (Leu)+(Glu),of the extracellular domain of CD154, both constructed as BamHI-XbaIfragments. The sense primer that fuses the two different truncatedextracellular domains to the 2H7scFv includes a BamHI site for cloning.The sense primer for the S4 form of the CD154 cDNA is designatedSEQUENCE ID NO: 535 or CD154BAM108 and encodes a 34 mer with thefollowing sequence: 5′-gtt gtc gga tcc aga aaa cag ctt tga aat gca a-3′,while the antisense primer is designated SEQUENCE ID NO: 536 or CD154XBAand encodes a 44 mer with the following sequence: 5′-gtt gtt tct aga ttatca ctc gag ttt gag taa gcc aaa gga cg-3′ (SEQ ID NO:536).

The oligonucleotide primers used in amplifying the long form (L2) of theCD154 extracellular domain encoding amino acids 48 (Arg)-261(Leu)+(Glu), were as follows: The sense primer designated CD154 BAM48(SEQUENCE ID NO:537) encoded a 35-mer with the following sequence:5′-gtt gtc gga tcc aag aag gtt gga caa gat aga ag-3′. The antisenseprimer designated or CD154XBA (SEQUENCE ID NO:538) encoded the 44-mer:5′-gtt gtt tct aga tta tca ctc gag ttt gag taa gcc aaa gga cg-3′. OtherPCR reaction conditions were identical to those used for amplifying the2H7 scFv (see Example 1). PCR fragments were purified by PCR quick kits(QIAGEN, San Diego, Calif.), eluted in 30 μl ddH₂O, and digested withBamHI and XbaI (Roche) restriction endonucleases in a 40 μl reactionvolume at 37° C. for 3 hours. Fragments were gel purified, purifiedusing QIAEX kits according to the manufacturer's instructions (QIAGEN),and ligated along with the 2H7 HindIII-BclI fragment into the pD18expression vector digested with HindIII+XbaI. Ligation reactions weretransformed into DH5-alpha chemically competent bacteria and plated ontoLB plates containing 100 μg/ml ampicillin. Transformants were grownovernight at 37° C., and isolated colonies used to inoculate 3 ml liquidcultures in Luria Broth containing 100 μg/ml ampicillin. Clones werescreened after mini-plasmid preparations (QIAGEN) for insertion of boththe 2H7 scFv and the CD154 extracellular domain fragments.

The 2H7scFv-CD154 construct cDNAs were subjected to cycle sequencing ona PE 9700 thermocycler using a 25-cycle program that includeddenaturating at 96° C., 10 seconds, annealing at 50° C. for 5 seconds,and extension at 60° C., for 4 minutes. The sequencing primers used werepD18 forward (SEQ ID NO:539: 5′-gtctatataagcagagctctggc-3′) and pD18reverse (SEQ ID NO:540: 5′-cgaggctgatcagcgagctctagca-3′) primers. Inaddition, an internal primer was used that had homology to the humanCD154 sequence (SEQ ID NO:541: 5′-ccgcaatttgaggattctgatcacc-3′).Sequencing reactions included primers at 3.2 pmol, approximately 200 ngDNA template, and 8 μl sequencing mix. Sequencing reactions wereperformed using the Big Dye Terminator Ready Sequencing Mix (PE-AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. Samples were subsequently purified using Centrisep columns(Princeton Separations, Adelphia, N.J.). The eluates were dried in aSavant speed-vacuum dryer, denatured in 20 μl template SuppressionReagent (ABI) at 95° C. for 2 minutes, and analyzed on an ABI 310Genetic Analyzer (PE-Applied Biosystems). The sequence was edited,translated, and analyzed using Vector Nti version 6.0 (Informax, NorthBethesda, Md.). The 2H7scFv-CD154 L2 cDNA sequence and predicted aminoacid sequence is presented in FIG. 7A, and 2H7scFv-CD154 S4 cDNAsequence and predicted amino acid sequence is presented in FIG. 7B.

The binding activity of the 2H7 scFv-CD154 fusion proteins (SEQ. ID NOs:691 and 693) to CD20 and CD40 simultaneously was determined by flowcytometry. The assay used CHO cell targets that express CD20. After a45-minute incubation of CD20 CHO cells with supernatants from cellstransfected with the 2H7 scFv-CD 154 expression plasmid, the CD20 CHOcells were washed twice and incubated with biotin-conjugated CD40-Igfusion protein in PBS/2% FBS. After 45 min, cells were washed twice andincubated with phycoerythrin (PE)-labeled strepavidin at 1:100 in PBS/2%FBS (Molecular Probes, Eugene Oreg.). After an additional 30 minincubation, cells were washed 2× and were analyzed by flow cytometry.The results show that the 2H7 scFv-CD154 molecule was able to bind toCD20 on the cell surface and to capture biotin-conjugated CD40 fromsolution (FIG. 8).

To determine the effect of the 2H7scFv-CD 154 on growth and viability ofB lymphoma and lymphoblastoid cell lines, cells were incubated with2H7scFv-CD154 L2 (SEQ. ID NO:691) for 12 hours and then examined forbinding of Annexin V. Binding of Annexin V was measured using aFITC-Annexin V kit (Immunotech, Marseille, France, Catalog #PN-IM2376).B cell lines were incubated in 1 ml cultures with dilutions ofconcentrated, dialyzed supernatants from cells expressing secreted formsof the 2H7scFv-CD154 fusion proteins. The results are presented in FIG.9.

The growth rate of the Ramos B lymphoma cell line in the presence of2H7scFv-CD154 was examined by uptake of ³H-thymidine for the last 6hours of a 24-hour culture. The effect of 2H7scFv-CD154 on cellproliferation is shown in FIG. 10.

Example 5 Construction and Characterization of CytoxB AntibodyDerivatives

CytoxB antibodies were prepared using 2H7 scFv-IgG polypeptide. The 2H7scFv (see Example 1) was linked to the human IgG1 Fc domain via analtered hinge domain (see FIG. 11). Cysteine residues in the hingeregion were substituted with serine residues by site-directedmutagenesis and other methods known in the art. The mutant hinge wasfused either to a wild-type Fc domain to create one construct,designated CytoxB-MHWTG1C, or was fused to a mutated Fc domain(CytoxB-MHMG1C) that had additional mutations introduced into the CH2domain. Amino acid residues in CH2 that are implicated in effectorfunction are illustrated in FIG. 11. Mutations of one or more of theseresidues may reduce FcR binding and mediation of effector functions. Inthis Example, the leucine residue 234 known in the art to be importantto Fc receptor binding, was mutated in the 2H7 scFv fusion protein,CytoxB-[MG1H/MG1C]. In another construct, the human IgG1 hinge regionwas substituted with a portion of the human IgA hinge, which was fusedto wild-type human Fc domain (CytoxB-IgAHWTHG1C). (See FIG. 11). Thismutated hinge region allows expression of a mixture of monomeric anddimeric molecules that retain functional properties of the human IgG1CH2 and CH3 domains. Synthetic, recombinant cDNA expression cassettesfor these molecules were constructed and polypeptides were expressed inCHODG44 cells according to methods described in Example 2.

Purified fusion protein derivatives of CytoxB-scFvIg molecules wereanalyzed by SDS-PAGE according to the methods described in Example 2.Polyacrylamide gels were run under non-reducing and reducing conditions.Two different molecule weight marker sets, BioRad prestained markers,(BioRad, Hercules, Calif.) and Novex Multimark molecular weight markerswere loaded onto each gel. The migration patterns of the differentconstructs and of Rituximab™ are presented in FIG. 12.

The ability of the different derivatives of CytoxB-scFvIg molecules tomediate ADCC was measured using the Bjab B lymphoma cells as the targetand freshly prepared human PBMCs as effector cells. (See Example 2).Effector to target ratios were varied as follows: 70:1, 35:1, and 18:1,with the number of Bjab cells per well remaining constant but the numberof PBMCs were varied. Bjab cells were labeled for 2 hours with ⁵¹Cr andaliquoted at a cell density of 5×10⁴ cells/well to each well offlat-bottom 96 well plates. Purified fusion proteins or rituximab wereadded at a concentration of 10 μg/ml to the various dilutions of PBMCs.Spontaneous release was measured without addition of PBMC or fusionprotein, and maximal release was measured by the addition of detergent(1% NP-40) to the appropriate wells. Reactions were incubated for 4hours, and 100 μl of culture supernatant was harvested to a Lumaplate(Packard Instruments) and allowed to dry overnight prior to counting cpmreleased. The results are presented in FIG. 13.

Complement dependent cytotoxicity (CDC) activity of the CytoxBderivatives was also measured. Reactions were performed essentially asdescribed in Example 2. The results are presented in FIG. 14 as percentof dead cells to total cells for each concentration of fusion protein.

Example 6 In Vivo Studies in Macaques

Initial in vivo studies with CytoxB derivatives have been performed innonhuman primates. FIG. 15 shows data characterizing the serum half-lifeof CytoxB in monkeys. Measurements were performed on serum samplesobtained from two different macaques (J99231 and K99334) after doses of6 mg/kg were administered to each monkey on the days indicated byarrows. For each sample, the level of 2H7scFvIg present was estimated bycomparison to a standard curve generated by binding of purifiedCytoxB-(MHWTG1C)-Ig fusion protein to CD20 CHO cells (see Example 2).The data are tabulated in the bottom panel of the FIG. 15.

The effect of CytoxB-(MHWTG1C)Ig fusion protein on levels of circulatingCD40+ cells in macaques was investigated. Complete blood counts wereperformed at each of the days indicated in FIG. 16. In addition, FACS(fluorescence activated cell sorter) assays were performed on peripheralblood lymphocytes using a CD40-specific fluorescein conjugated antibodyto detect B cells among the cell population. The percentage of positivecells was then used to calculate the number of B cells in the originalsamples. The data are graphed as thousands of B cells per microliter ofblood measured at the days indicated after injection (FIG. 16).

Example 7 Construction and Expression of an Anti-CD19 scFv-Ig FusionProtein

An anti-CD19 scFv-Ig fusion protein was constructed, transfected intoeukaryotic cells, and expressed according to methods presented inExamples 1, 2, and 5 and standard in the art. The variable heavy chainregions and variable light chain regions were cloned from RNA isolatedfrom hybridoma cells producing antibody HD37, which specifically bindsto CD19. Expression levels of a HD37scFv-IgAHWTG1C and aHD37scFv-IgMHWTG1C were measured and compared to a standard curvegenerated using purified HD37 scFvIg. The results are presented in FIG.17.

Example 8 Construction and Expression of an Anti-L6 scFv-Ig FusionProtein

An scFv-Ig fusion protein was constructed using variable regions derivedfrom an anti-carcinoma monoclonal antibody, L6. The fusion protein wasconstructed, transfected into eukaryotic cells, and expressed accordingto methods presented in Examples 1, 2, and 5 and standard in the art.Expression levels of L6 scFv-IgAH WCH2 CH3 and L6 scFv-(SSS-S)H WCH2WCH3 were measured and compared to a standard curve generated usingpurified L6 scFvIg. The results are presented in FIG. 18.

Example 9 Characterization of Various scFv-Ig Fusion Proteins

In addition to the scFv-Ig fusion protein already described, G28-1(anti-CD37) scFv-Ig fusion proteins were prepared essentially asdescribed in Examples 1 and 5. The variable regions of the heavy andlight chains were cloned according to methods known in the art. ADCCactivity of 2H7-MHWTG1C, 2H7-IgAHWTG1C, G28-1-MHWTG1C, G28-1 IgAHWTG1C,HD37-MHWTG1C, and HD37-IgAHWTG1C was determined according to methodsdescribed above (see Example 2). Results are presented in FIG. 19. ADCCactivity of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C was measured using the2981 human lung carcinoma cell line. The results are presented in FIG.20. The murine L6 monoclonal antibody is known not to exhibit ADCCactivity.

The purified proteins were analyzed by SDS-PAGE under reducing andnon-reducing conditions. Samples were prepared and gels run essentiallyas described in Examples 2 and 5. The results for the L6 and 2H7 scFv-Igfusion proteins are presented in FIG. 21 and the results for the G28-1and HD37 scFv-Ig fusion proteins are presented in FIG. 22.

Example 10 Construction and Expression of Anti-CD20 scFv-Llama Ig FusionProteins

This Example illustrates the cloning of llama IgG1, IgG2, and IgG3constant region domains and the construction of immunoglobulin fusionproteins with each of the three constant regions and anti-CD20 scFv.

The constant regions of llama IgG1, IgG2, and IgG3 immunoglobulins werecloned and inserted into mammalian vector constructs containing ananti-CD20 single chain Fv, 2H7 scFv. Total RNA was isolated fromperipheral blood mononuclear cells (PBMC) from llama blood (Triple JFarms, Bellingham, Wash.) by lysing the lymphocytes in TRIzol®(Invitrogen Life Technologies, Carlsbad, Calif.) according to themanufacturer's instructions. One microgram (1 μg) of total RNA was usedas template to prepare cDNA by reverse transcription. The RNA and 200 ngrandom primers were combined and denatured at 72° C. for 10 minutesprior to addition of enzyme. Superscript II reverse transcriptase(Invitrogen Life Technologies) was added to the RNA plus primer mixturein a total volume of 25 μl in the presence of 5× second strand bufferand 0.1 M DTT provided with the enzyme. The reverse transcriptionreaction was allowed to proceed at 42° C. for one hour. The cDNA wasamplified by PCR using sequence specific primers. The 5′ primers weredesigned according to published sequences for the V_(HH) and V_(H)domains of camelids. The 3′ primer, which was used to amplify all threeisotypes, was designed using mammalian CH3 domain sequences as a guide.The following specific primers were used. The Bcl and XbaI sites areindicated by underlined italicized sequences.

5′ primer for llama IgG1 constant region    LLG1-5′bgl: (SEQ ID NO: 542)   5′-gtt gt t gat c aa gaa cca cat gga gga tgc acg tg-3′ 5′ primer forllama IgG2 constant region    LLG2-5′bgl: (SEQ ID NO: 543)    5′-gtt gtt gat c aa gaa ccc aag aca cca aaa cc-3′ 5′ primer for llama IgG3constant region    LLG3-5′bgl: (SEQ ID NO: 544)    5′-gtt gt t gat c aagcg cac cac agc gaa gac ccc-3′ 3′ primer for llama IgG1, IgG2, and IgG3constant regions    LLG123-3′X: (SEQ ID NO: 545)    5′-gtt gtt tct aga tta cta ttt acc cga aga ctg ggt gat gga-3′

PCR fragments of the expected size were cloned into TOPO® cloningvectors (Invitrogen Life Technologies) and then were sequenced. Thesense sequencing primer, LLseqsense, had the sequence 5′-ctg aga tcg agttca gct g-3′ (SEQ ID NO:546), and the antisense primer, LLseqAS, had thesequence 5′-cct cct ttg gct ttg tct c-3′ (SEQ ID NO:547). Sequencing wasperformed as described in Example 1. FIG. 23 compares the amino acidsequence of the three isotype llama constant regions containing thehinge, CH2, and CH3 domains with the amino acid sequence of human IgG1hinge, CH2, and CH3 domains.

After verifying the sequence, the amplified PCR products were digestedwith restriction enzymes BclI and Xba1 to create compatible restrictionsites. The digested fragments were then gel-purified, and the DNA waseluted using a QIAquick Gel Extraction Kit (QIAGEN, Valencia, Calif.).The 2H7scFv-Ig pD18 mammalian expression vector construct (see Example2) was digested with BclI and XbaI to remove the human IgG hinge, CH2,and CH3 domains. The pD18 vector is a modified derivative of pcDNA3 thatcontains an attenuated DHFR gene, which serves as a selectable markerfor mammalian expression (Hayden et al., Tissue Antigens 48:242-54(1996)). The purified llama IgG1, IgG2, and IgG3 constant region PCRproducts were ligated by T4 DNA ligase (Roche Molecular Biochemicals,Indianapolis, Ind.) into the double-digested 2H7 scFv-pD18 vector atroom temperature overnight according to the manufacturer's instructions.After ligation, the ligation products were transformed into E. coli DH5abacteria (BD Biosciences, Palo Alto, Calif.) and plated according tostandard molecular biology procedures and manufacturer's instructions.Isolated colonies were chosen to screen for transformants containing thecorrect inserts.

For expression of the encoded polypeptides, plasmid DNA from positiveclones was transiently transfected into COS-7 cells using DEAE-dextran(Hayden et al., Ther Immunol. 1:3-15 (1994)). COS-7 cells were seeded atapproximately 3×10⁶ cells per 150 mm plate and grown overnight until thecells were about 75% confluent. Cells were then washed once withserum-free DMEM (Invitrogen Life Technologies, Grand Island, N.Y.).Transfection supernatant (10 ml) containing 400 μg/ml DEAE-dextran, 0.1mM chloroquine, and 5 μg/ml of the DNA constructs were added to thecells, which were then incubated at 37° C. for 3-4 hrs. Afterincubation, cells were pulsed with 10 ml of 10% dimethyl sulfoxide(DMSO) in 1×PBS at room temperature for 2 minutes. Cells were thenplaced back into fully supplemented DMEM/10% FBS (1% L-glutamine, 1%penicillin/streptomycin, 1% sodium pyruvate, 1% MEM essential aminoacids) (Invitrogen Life Technologies). After 24 hours, the media wasreplaced with serum-free fully supplemented DMEM (Invitrogen LifeTechnologies), and the cells were maintained up to 21 days with mediachanges every 3-4 days.

Ig-fusion proteins were purified by passing COS cell culturesupernatants through Protein A Agarose (Repligen, Cambridge, Mass.)columns. After application of the culture supernatant, the Protein Acolumns were then washed with 1×PBS (Invitrogen Life Technologies).Bound Ig-fusion proteins were eluted with 0.1 M citric acid (pH 2.8),and the collected fractions were immediately neutralized with Tris base(pH 10.85). The fractions containing protein were identified bymeasuring the optical density (A₂₈₀) and then were pooled, dialyzedagainst 1×PBS, (Invitrogen Life Technologies) and filtered through a 0.2μm filter.

The purified Ig-fusion proteins were analyzed by SDS-PAGE. Aliquots of2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7 scFv-llama IgG3, andRituxan® (Rituximab, anti-CD20 antibody, Genentech, Inc. and IDECPharmaceuticals Corp.) (5 μg protein) were combined with 25 μl 2×NuPAGE® SDS Sample Buffer (Invitrogen Life Technologies) (non-reducedsamples). Samples of each protein were also prepared in reducing samplebuffer containing 5% 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.).Molecular weight markers (Invitrogen Life Technologies) were applied tothe gels in non-reducing buffer only. The proteins were fractionated onNuPAGE® 10% Bis-Tris gels (Invitrogen Life Technologies). Afterelectrophoresis (approximately 1 hour), the gels were washed threetimes, five minutes each, with Distilled Water (Invitrogen LifeTechnologies) and then stained in 50 ml Bio-Safe Coommassie Stain(BioRad, Hercules, Calif.) overnight at room temperature. After a washin Distilled Water, the gels were photographed. The migration pattern ofeach Ig-fusion protein is presented in FIG. 24.

The ability of the 2H7 scFv-llama Ig fusion proteins to bind to cellsexpressing CD20 was demonstrated by flow cytometry. Serial dilutionsstarting at 25 μg/ml of purified 2H7 scFv-llama IgG1, 2H7 scFv-llamaIgG2, and 2H7 scFv-llama IgG3 were prepared and incubated withCD20-transfected (CD20+) CHO cells (from the laboratory of Dr. S. Skov,Institute of Medical Microbiology and Immunology, Copenhagen Denmark in1% FBS 1×PBS media (Invitrogen Life Technologies) for one hour on ice.After the incubation, the cells were then centrifuged and washed with 1%FBS in 1×PBS. To detect bound 2H7 scFv-llama Ig, the cells wereincubated for one hour on ice with fluorescein-conjugated goatanti-camelid IgG (heavy and light chain) (1:100) (Triple J Farms). Thecells were then centrifuged and resuspended in 1% FBS-1×PBS and analyzedusing a Coulter Epics XL cell sorter (Beckman Coulter, Miami, Fla.). Thedata (percent of maximum brightness) are presented in FIG. 25.

Example 11 Effector Function of Anti-CD20 scFv-Llama Ig Fusion Proteins

This Example demonstrates the ability of anti-CD20 llama IgG1, IgG2, andIgG3 fusion proteins to mediate complement dependent cytotoxicity (CDC)and antibody dependent cell-mediated cytotoxicity (ADCC).

The ability of the 2H7 scFv-llama IgG fusion proteins to kill CD20positive cells in the presence of complement was tested using the BJABhuman B cell line. Rabbit complement was obtained from 3-4 week oldrabbits (Pel-Freez, Brown Deer, Wis.). BJAB cells (2×10⁶ cells/ml) werecombined with rabbit complement (final dilution 1:10) and purified 2H7Ig fusion proteins. 2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7scFv-llama IgG3, and 2H7 scFv-human IgG1 wild type hinge-CH2-CH3)(Example 1) were added at 1:3 serial dilutions beginning at aconcentration of 30 μg/ml. After one hour at 37° C., cell viability wasdetermined by counting live and dead cells by trypan blue exclusion(0.4%) (Invitrogen Life Technologies) using a hemacytometer(Bright-line, Horsham, Pa.). The percent killing was calculated bydividing the number of dead cells by the number of total cells(dead+live cells). The data presented in FIG. 26 show that all Ig fusionproteins had CDC activity.

The ADCC activity of the 2H7 scFv-llama IgG fusion proteins wasdetermined using BJAB cells as target cells and human or llamaperipheral blood mononuclear cells (PBMC) as effector cells. BJAB cellswere pre-incubated for approximately 2 hours with ⁵¹Cr (100 μCi)(Amersham Biosciences, Piscataway, N.J.) in fully supplemented IMDM(Invitrogen Life Technologies) containing 15% FBS. The cells were mixedintermittently during the pre-incubation period. Fresh, resting humanPBMC were purified from whole blood using Lymphocyte Separation Media(LSM) (ICN Pharmaceuticals, New York, N.Y.). PBMC were combined withlabeled BJAB cells (5×10⁴ cells per well of 96 well tissue cultureplate) at ratios of 25:1, 50:1, and 100:1. To each combination was added10 μg/ml of purified 2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7scFv-llama IgG3, Rituximab, or no anti-CD20 antibody. The mixtures wereincubated for 6 hours at 37° C. Supernatant from each reactioncontaining ⁵¹Cr released from lysed cells was collected onto aLumaPlate-96 filter plate (Packard, Meriden, Conn.), which was driedovernight. The amount of ⁵¹Cr was measured by a TopCount NXT platereader (Packard). FIG. 27 shows that the 2H7 scFv-llama IgG2 fusionprotein was the most effective llama fusion protein in mediating ADCC.Each data point represents the average measurement of triplicate wells.

ADCC activity was affected by the source of effector cells. Llama PBMCwere isolated from llama blood (Triple J Farms) using LSM. Llamaeffector cells were added at the same ratios to BJAB target cells asdescribed for the ADCC assay using human effector cells. The cells werecombined with 10 μg/ml of purified 2H7 scFv-llama IgG1, 2H7 scFv-llamaIgG2, 2H7 scFv-llama IgG3, Rituximab, or no anti-CD20 antibody. Theresults are presented in FIG. 28.

Example 12 Construction and Characterization of scFv Ig Fusion ProteinsExpressed on the Cell Surface

This Example describes a retroviral transfection system for ectopicsurface expression of genetically engineered cell surface receptorscomposed of scFvs that bind costimulatory receptors. The Example alsodemonstrates the effector function of these various scFv Ig fusionproteins expressed on the surface of target cells.

The heavy and light chain variable regions were cloned from murinemonoclonal antibodies specific for various costimulatory receptors, andsingle chain Fv constructs were prepared essentially as described inExample 1. Antibodies included 2H7, anti-human CD20; 40.2.220,anti-human CD40; 2E12, anti-human CD28; 10A8, anti-human CD152(anti-CTLA-4); and 500A2, anti-murine CD3. The heavy chain and lightchain variable regions of each antibody were cloned according tostandard methods for cloning immunoglobulin genes and as described inExample 1. Single chain Fv constructs were prepared as described inExample 1 by inserting a nucleotide sequence encoding a (gly₄ser)₃ (SEQID NO:529) peptide linker between the VL region nucleotide sequence of40.2.220, 2E12, 10A8, and 500A2, respectively (SEQ ID NOs:31, 37 and 43,respectively) and the VH region nucleotide sequence of 40.2.220, 2E12,10A8, and 500A2, respectively (SEQ ID NOs:33, 39 and 45, respectively).The polypeptide sequence for VL of 40.2.220, 2E12, 10A8, and 500A2 areset forth in SEQ ID NOs:32, 38 and 44, respectively, and the polypeptidesequence for VH of 40.2.220, 2E12, 10A8, and 500A2 are set forth in SEQID NOs:30, 40 and 46, respectively. Each scFv polynucleotide (SEQ IDNOs:36, 42 and 47 for 40.2.220, 2E12, 10A8, and 500A2, respectively) wasthen fused to human IgG1 mutant hinge (CCC→SSS) and mutant CH2 (prolineto serine mutation at residue 238 (238 numbering according to EUnomenclature, Ward et al., 1995 Therap. Immunol. 2:77-94; residue 251according to Kabat et al.) and wild type CH3 domains according to themethods described in Example 5 and 11. Each scFv mutant IgG1 fusionpolynucleotide sequence was then fused in frame to sequences encodingthe transmembrane domain and cytoplasmic tail of human CD80 (SEQ IDNO:29), such that when the fusion protein was expressed in thetransfected cell, CD80 provided an anchor for surface expression of thescFv Ig fusion protein. cDNAs encoding the scFv-IgG-CD80 fusion proteins(SEQ ID NOs:49, 51, 53 and 55 for 40.2.220-, 2E12-, 10A8-, and500A2-scFv-IgG-CD80, respectively) were inserted into the retroviralvector pLNCX (BD Biosciences Clontech, Palo Alto, Calif.) according tostandard molecular biology procedures and vendor instructions. ThescFv-Ig-CD80 cDNA was inserted between the 5′LTR-neomycin resistancegene-CMV promoter sequences and the 3′LTR sequence. The retroviralconstructs were transfected into Reh, an acute lymphocytic leukemia cellline (ATCC CRL-8286). Transfected cells were screened to select clonesthat were expressing scFv-Ig fusion proteins on the cell surface.

CDC and ADCC assays were performed with the transfected Reh cells todetermine if expression of the scFv-Ig polypeptides on the cell surfaceaugmented effector cell function. Reh cells expressing anti-human CD152scFv-mutant IgG-CD80 (SEQ ID NO:56); Reh anti-human CD28 scFv-mutantIgG-CD80 (SEQ ID NO:52);; Reh anti-human CD40 scFv-mutant IgG-CD80 (SEQID NO50:); Reh anti-human CD20 scFv-mutant IgG-CD80 (SEQ ID NO:_) werecombined with human PBMC (see Example 11) and rabbit complement (10μg/ml) for one hour at 37° C. Untransfected Reh cells were included as acontrol. Viability of the cells was determined by trypan blue exclusion,and the percent of killed cells was calculated (see Example 11). FIG. 29shows the effectiveness of the scFv-IgG-CD80 fusion proteins whenexpressed on the cell surface of tumor cells to mediate complementdependent cytotoxicity.

The same transfected Reh cells tested in the CDC assay plus Reh cellstransfected with the polynucleotide construct that encodes anti-murineCD3-scFv-Ig-CD80 (SEQ ID NO:110) were analyzed for ADCC activity (seeExample 11). Untransfected and transfected Reh cells were pre-labeledwith ⁵¹Cr (100 μCi) (Amersham) for two hours at 37° C. Human PBMC servedas effector cells and were added to the Reh target cells (5×10⁴ cellsper well of 96 well plate) at ratios of 5:1, 2.5:1, and 1.25:1. Afterfive hours at 37° C., culture supernatants were harvested and analyzedas described in Example 11. Percent specific killing was calculatedaccording to the following equation: ((experiment release minusspontaneous release)/(maximum release minus spontaneous release))×100.The data are presented in FIG. 30. Each data point represents theaverage of quadruplicate samples.

Using the same procedures described above, the same results with otherbinding domains were obtained using the following monoclonal antibodiesmonoclonal antibodies as sources of sFv: for CD20, 1F5 (Genbank AY058907 and AY058906); for CD40, 2.36 and G28.5; for CD28, 9.3.

Cell surface expression of antibody binding domains is accomplished byfusing antibody scFvs to IgA hinge and constant regions and IgEhinge-acting region, i.e., IgE CH2, and constant regions.Polynucleotides encoding an anti-4-1BB scFv, 5B9 (anti-human 4-1BB)scFv, and 2e12 (anti-human CD40) fused to IgAH IgA T4 (four terminal CH3residues deleted) fused to the CD80 transmembrane and cytoplasmicdomains and IgE Fc regions are shown in SEQ ID NOs:177, 181, 179 and183. The encoded polypeptides are shown in SEQ ID NOs:178, 182, 180 and184.

Example 13 Construction and Sequence of Human Ig Hinge-CH2-CH3 Mutantsand 2H7 Variable Region Mutants

This Example describes construction of scFv fusion proteins containingmutant human IgG1 and IgA constant regions. This Example also describesconstruction of a 2H7 scFv mutant with a single point mutation in thevariable heavy chain region. Mutations were introduced into variable andconstant region domains according to methods described herein and knownin the molecular biology arts. FIG. 31 presents nomenclature for the Igconstant region constructs.

The human IgG1 hinge region of the 2H7 scFv human IgG1 hinge-CH2-CH3fusion proteins was mutated to substitute cysteine residues that in awhole immunoglobulin are involved in forming disulfide bonds between twoheavy chain molecules. One mutant, 2H7 scFv fused to a human IgG1 hingeregion in which all three cysteine residues were mutated to serineresidues (MTH (SSS)), was prepared as described in Example 5 (designatedin Example 5 as CytoxB-MHWTG1C (includes wild type IgG1 CH2 and CH3domains)) (now referred to as 2H7 scFv MTH (SSS) WTCH2CH3) and comprisesthe polynucleotide sequence SEQ ID NO:57 encoding the polypeptide as setforth in SEQ ID NO:58. The polynucleotide sequence encoding this mutant(SEQ ID NO:57) was used as a template to create mutant hinge regions inwhich the first two cysteine residues were substituted with serineresidues (IgG MTH (SSC)). An oligonucleotide was designed to substitutethe third serine residue with a cysteine and had the following sequence:5′-gtt gtt gat cag gag ccc aaa tct tct gac aaa act cac aca tct cca ccgtgc cca gca cct g-3′ (HuIgGMHncs3, SEQ ID NO:548). A second mutant wasprepared in which the mutant hinge had serine residues substituting thefirst and third cysteine residues (IgG MTH (SCS)). The sequence of theoligonucleotide to create this mutant was as follows: 5′-gtt gtt gat caggag ccc aaa tct tct gac aaa act cac aca tgc cca ccg-3′ (HuIgGMHncs2, SEQID NO:549). A third mutant was prepared with cysteine residuessubstituted at the second and third positions (IgG MTH (CSS)), alsousing the IgG MTH (SSS) mutant as template, and an oligonucleotidehaving the sequence, 5′-gtt gtt gat cag gag ccc aaa tct tgt gac aaa actcac-3′ (HuIgGMHncs1, SEQ ID NO:550).

The oligonucleotides introducing the mutations into the hinge regionwere combined with template and a 3′ oligonucleotide containing an XbaIsite (underlined and italicized) (5′-gtt gtt tct aga tca ttt acc cgg agacag gga gag get ctt ctg cgt gta g-3′ (SEQ ID NO:551)) to amplify themutant hinge-wild type (WT)-CH2-CH3 sequences by PCR. The IgG MTH CSSand IgG MTH SCS mutant sequences were amplified for 25 cycles with adenaturation profile of 94° C., annealing at 52° C. for 30 seconds, andextension at 72° C. for 30 seconds. The IgG MTH SSC mutant sequence wasamplified under slightly different conditions: denaturation profile of94° C., annealing at 45° C. for 30 seconds, and extension at 72° C. for45 seconds. The amplified polynucleotides were inserted into the TOPO®cloning vector (Invitrogen Life Technologies) and then were sequenced asdescribed in Example 1 to confirm the presence of the mutation. pD18vector containing 2H7 scFv was digested to remove the constant regionsequences essentially as described in Example 10. The mutant hinge-wildtype CH2-CH3 regions were inserted in frame into the digested vector DNAto obtain vectors comprising 2H7 scFv MTH (CSS) WTCH2CH3 encoding DNA(SEQ ID NO:134); 2H7 scFv MTH (SCS) WTCH2CH3 encoding DNA (SEQ IDNO:136); and 2H7 scFv MTH (SSC) WTCH2CH3 encoding DNA (SEQ ID NO:138).

A mutation of leucine to serine at position 11 in the first frameworkregion of the heavy chain variable region (numbering according to Kabatet al., Sequences of Proteins of Immunological Interest, 5^(th) ed.Bethesda, Md.: Public Health Service, National Institutes of Health(1991)) was introduced into the 2H7 scFv MTH (SSS) WTCH2CH3 fusionprotein (SEQ ID NO:58). The wild type leucine residue was substitutedwith serine by site-directed mutagenesis using the oligonucleotideVhser11: 5′-gga ggt ggg agc tct cag gct tat cta cag cag tct ggg gct gagtcg gtg agg cc-3′ (SEQ ID NO:552)(this sequence, or an amino acidsequence that it encodes, may be optionally excluded from particularclaimed embodiments of the instant invention). The 3′-primer for PCR washuIgG1-3′ having the sequence 5′-gtc tct aga cta tca ttt acc cgg agacag-3′ (SEQ ID NO:553) (XbaI site underlined and italicized). After PCRamplification, the fragments were inserted into the TOPO® cloning vectorand sequenced to confirm the presence of the VH11 leucine to serinemutation. The 2H7 scFv-IgG MTH (SSS) WTCH2CH3 encoding DNA was shuttledinto the PSL1180 cloning vector (Pharmacia Biotech, Inc., Piscataway,N.J.). The construct PSL1180-2H7 scFv-IgG MTH (SSS) WTCH2CH3 wasdigested with Sac and XbaI to remove the wild type VH domain and thehinge and CH2 and CH3 domains. The PCR product comprising the VH11mutant was digested with Sac and XbaI and then inserted into thedigested PSL1180 construct according to standard molecular biologyprocedures. The construct was then digested with Hind III and XbaI, andinserted into the mammalian expression vector pD18 (see methodsdescribed in Example 1 and Example 10). The mutant is designated 2H7scFv VH11SER IgG MTH (SSS) WTCH2CH3 (FIG. 31). The polynucleotidesequence is provided in SEQ ID NO:369, and the encoded polypeptidesequence is provided in SEQ ID NO:370 (these sequences may be optionallyexcluded from particular claimed embodiments of the instant invention).

Four constructs containing IgA constant region domains were prepared.One construct contained wild type IgA hinge fused to human IgG1 CH2 andCH3 (IgAH IgG WTCH2CH3) (FIG. 31). Sequential PCR amplifications wereperformed to substitute the human IgG1 hinge of the 2H7 scFv constructwith nucleotide sequences encoding the IgA hinge. The 5′ oligonucleotideprimer (huIgA/Gchim5) for the first PCR reaction had the sequence,5′-cca tct ccc tca act cca cct acc cca tct ccc tca tgc gca cct gaa ctcctg-3′ (SEQ ID NO:554). The primer (huIgAhg-5′) for the second PCRreaction to add more IgA specific hinge sequence and add a BclIrestriction enzyme site (italicized and underlined) had the sequence,5′-gtt gat cag cca gtt ccc tca act cca cct acc cca tct ccc caa ct-3′(SEQ ID NO:555). The 3′ primer for both amplification steps washuIgG1-3′ having the sequence, 5′-gtc tct aga cta tca ttt acc cgg agacag-3′ (SEQ ID NO:556). The sequence of the PCR product was confirmed byTOPO® cloning as described above. The gel-purified fragment was digestedwith BclI and XbaI and then inserted into the 2H7 scFv-pD18 vector thathad been digested BclI and XbaI to remove all the IgG1 constant regiondomains. Ligation was performed as described in Example 10 to provide amammalian expression vector comprising the nucleotide sequence (SEQ IDNO:59) encoding a 2H7 scFv IgA hinge-IgG1 CH2-CH3 polypeptide (SEQ IDNO:60).

A second pD18 mammalian expression vector was constructed that had apolynucleotide sequence (SEQ ID NO:61) that encoded a 2H7 scFv fused towild type IgA hinge, CH2, and CH3 domains (SEQ ID NO:62). Human IgAconstant regions sequences were obtained by using random primers toreverse transcribe total RNA isolated from human tonsil followed by PCRamplification of the cDNA using sequence specific primers, essentiallyas described in Example 10. Human IgA hinge-CH2-CH3 nucleotide sequence(SEQ ID NO:63) encoding the IgA-CH2-CH3 polypeptide (IgAH IgACH2CH3,FIG. 31) (SEQ ID NO:64) was amplified using the 5′ oligonucleotidehuIgAhg-5′ (SEQ ID NO:555 and a 3′ oligonucleotide huIgA3′ having thesequence, 5′-gtt gtt tct aga tta tca gta gca ggt gcc gtc cac ctc cgc catgac aac-3′ (SEQ ID NO:557). Secretion of a 2H7-IgA hinge-IgA CH2-CH3polypeptide from transfected mammalian cells required co-expression ofhuman J chain that covalently binds to two IgA CH3 domains via disulfidebonds. Total RNA was isolated from tonsil B cells and was reversedtranscribed to generate cDNA as described above. PCR amplification ofthe nucleotide sequence encoding the J chain was performed with J chainspecific primers. The 5′ PCR primer, HUJCH5n1, had the sequence, 5′-gttgtt aga tct caa gaa gat gaa agg att gtt ctt-3′ (SEQ ID NO:558), andsequence of the 3′ primer, HUJCH3, was 5′-gtt gtt tct aga tta gtc aggata gca ggc atc tgg-3′ (SEQ ID NO:559). The cDNA was cloned into TOPO®for sequencing as described in Example 10. J chain encoding cDNA (SEQ IDNO:65) was then inserted into pD18 and pcDNA3-Hygro (+) (Invitrogen LifeTechnology) vectors for co-transfection with 2H7 scFv IgA hinge-CH2-CH3constructs. The J chain has the predicted amino acid sequence set forthin SEQ ID NO:66.

Secretion of an scFv IgA constant region construct in the absence of Jchain was accomplished by engineering a truncated CH3 domain with adeletion of the four carboxy terminal amino acids (GTCY, SEQ ID NO:67)(IgAH IgA-T4, FIG. 31), which include a cysteine residue that forms adisulfide bond with the J chain. The IgA hinge-CH2-CH3 nucleotidesequence containing the deletion in CH3 (SEQ ID NO:68) was preparedusing a 5′ PCR primer (huIgAhg-5′) having the sequence 5′-gtt gtt gatcag cca gtt ccc tca act cca cct acc cca tct ccc tca act-3′ (SEQ IDNO:561) (BclI site is underlined and italicized), and a 3′ PCR primer(HUIGA3T1) having the sequence 5′-gtt gtt tct aga tta tca gtc cac ctccgc cat gac aac aga cac-3′ (SEQ ID NO:562). This mutated IgA constantregion nucleotide sequence was inserted into a 2H7 scFv pD18 vector asdescribed for the generation of the previous 2H7 scFv-Ig constructs (seeExample 1 and this Example) that comprises the polynucleotide sequence(SEQ ID NO:70) encoding a 2H7 IgAH IgA-T4 polynucleotide (SEQ ID NO:71).

A fourth construct was prepared that encoded a 2H7 scFv-IgA constantregion fusion protein with a deletion of 14 additional amino acids, mostof which are hydrophobic residues, from the carboxy terminus of IgA CH3.The 2H7 scFv-IgAH IgA-T4 encoding polynucleotide was used as template toengineer a deletion of the nucleotide sequence encoding PTHVNVSVVMAEVD(SEQ ID NO:72). The 5′ oligonucleotide primer had the sequence 5′-gttgtt gat cag cca gtt ccc tca act cca cct acc cca tct ccc tca act-3′ (SEQID NO:564) (BclI site shown as underlined and italicized). The 3′oligonucleotide sequence was 5′-gtt gtt tct aga tta tca ttt acc cgc caagcg gtc gat ggt ctt-3′ (SEQ ID NO:565). The deleted IgA CH3 region wasamplified by using the above oligonucleotides to amplify the IgAconstant region from RNA isolated from human tonsil such that the cDNAcontained the deleted carboxyl terminal encoding region for the 18 aminoacids. The IgAH IgA-T18 constant region was inserted into a 2H7 scFvpD18 vector that comprises the polynucleotide sequence (SEQ ID NO:75)encoding a 2H7 IgAH IgA-T18 polynucleotide (SEQ ID NO:76) as describedabove.

Example 14 Effector Function of CTLA-4 IgG Fusion Proteins

The Example compares the effector functions of CTLA-4 Ig fusion proteinsin CDC and ADCC assays.

Two CTLA-4 IgG fusion proteins were constructed. One fusion proteincomprises the extracellular domain of CTLA-4 fused to human IgG1 wildtype hinge, CH2, and CH3 domains and is designated CTLA-4 IgG WTH (CCC)WTCH2CH3 (SEQ ID NO78). A pD18 mammalian expression vector comprising apolynucleotide sequence encoding CTLA-4 IgG WTH (CCC) WTCH2CH3 (SEQ IDNO:77) was prepared by fusing in frame the nucleotide sequence encodingthe extracellular domain of CTLA-4 (SEQ ID NO:83) (see U.S. Pat. No.5,844,095) to the nucleotide sequence encoding IgG WTH (CCC) WTCH2CH3(SEQ ID NO:1) according to the methods described in Examples 1 and 10.The extracellular domain nucleotide sequence also comprises a BclIrestriction enzyme site at the 3′ end, and a leader peptide nucleotidesequence (SEQ ID NO:79) that encodes an oncoM leader peptide (SEQ IDNO:80). A second CTLA-4 IgG fusion protein, designated CTLA-4 IgG MTH(SSS) MTCH2WTCH3, contained the extracellular domain of CTLA-4 (plus theoncoM leader peptide sequence) fused to a mutant IgG hinge in which allthree cysteine residues were replaced with serine residues. The hingeregion was fused to a mutant IgG1 CH2 domain that had a mutation atisotype position 238 (EU numbering, Ward et al., supra, (position 251using numbering according to Kabat et al., supra; position 209 wherenumbering commences with first residue of IgG1 CH1; i.e., PAPELLDGPS(SEQ ID NO:566) of wild type IgG1 CH2 is modified to PAPELLDGSS (SEQ IDNO:567)), which was fused to IgG1 wild type CH3 (U.S. Pat. No.5,844,095). The CTLA-4 IgG MTH (SSS) MTCH2WTCH3 polynucleotide comprisesthe nucleotide sequence in SEQ ID NO:85 and the deduced amino acidsequence comprises the sequence provided in SEQ ID NO:86. CTLA-4 fusionproteins were also prepared using CTLA-4 extracellular membrane encodingsequences without the leader peptide (SEQ ID NO:84).

To measure CDC activity, purified CTLA-4 IgG WTH (CCC) WTCH2CH3 (2μg/ml) or CTLA-4 IgG MTH (SSS) MTCH2WTCH3 (2 μg/ml) was added to Rehcells (see Example 12) and to Reh cells transfected with thecostimulatory molecule CD80 such that CD80 was expressed on the cellsurface (Reh CD80.10; see Doty et al., 1998 J. Immunol. 161:2700; Dotyet al., 1996 J. Immunol. 157:3270), in the presence or absence of rabbitcomplement (10 μg/ml). Purified CTLA Ig fusion proteins were preparedfrom culture supernatants of transiently transfected COS cells accordingto methods described in Example 10. The assays were performedessentially as described in Example 11 and 12. The data presented inFIG. 32 show that only CD80-transfected Reh cells were killed in thepresence of complement and CTLA-4 IgG WTH (CCC) WTCH2CH3 fusion protein.

The purified CTLA-4 Ig fusion proteins were also tested in ADCC assays.Human PBMC, serving as effector cells, were added to Reh or Reh CD80.1target cells at a ratio of 1.25:1, 2.5:1, 5.0:1, and 10:1. Cells werelabeled and the assays performed essentially as described in Examples 11and 12. The results are presented in FIG. 33. Each data point representsthe average of four independent culture wells at each effector:targetcell ratio. The data show that only CTLA-4 IgG WTH (CCC) WTCH2CH3mediated significant ADCC of Reh CD80.10 cells.

Example 15 Effector Function of CTLA-4 IgA Fusion Proteins

CTLA-4 IgA fusion proteins are prepared as described for the IgG fusionproteins (see Examples 1, 13, and 14). CTLA-4 extracellular domainnucleotide sequence (SEQ ID NO:84) is fused in open reading frame tonucleotides encoding IgAH IgACH2CH3 (SEQ ID NO:63) to provide thenucleotide sequence (SEQ ID NO:87) encoding a CTLA-4 IgAH IgACH2CH3fusion protein (SEQ ID NO:88). The fusion protein is transientlyexpressed in COS cells (see Example 10) or stably expressed in CHO cells(see Example 1). Secretion of the CTLA-4 IgAH IgACH2CH3 fusion proteinrequires co-transfection with a construct containing a polynucleotidesequence (SEQ ID NO:65) that encodes human J chain (SEQ ID NO:66). TheCTLA-4 IgAH IgACH2CH3 fusion protein is isolated as described inExamples 10 and 14. To express a CTLA-4 IgA construct without thepresence of J chain, a CTLA-4 IgAH IgA-T4 construct is prepared andtransfected into mammalian cells. In a similar manner as described forthe CTLA-4 extracellular fragment fused to wild type IgA hinge-CH2CH3,the CTLA-4 extracellular domain nucleotide sequence (SEQ ID NO:84) isfused in open reading frame to a nucleotide sequence (SEQ ID NO:68)encoding a IgAH IgA-T4 polypeptide (SEQ ID NO:69) to provide anucleotide sequence comprising SEQ ID NO:89 encoding a CTLA-4 IgAHIgA-T4 polypeptide (SEQ ID NO:90). Effector function of each constructis evaluated by CDC and ADCC as described in Example 14.

Example 16 Binding of Anti-CD20 scFv Human Ig Fusion Proteins to CHOCells Expressing CD20

This Example describes binding of 2H7 scFv Ig fusion proteins to CHOcells that express CD20. The analysis was performed by flow cytometry.Culture supernatants were collected from transiently transfected COScells expressing 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:28); 2H7scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID NO:135); 2H7 scFv IgG MTH (SCS)WTCH2CH3 (SEQ ID NO:137); and 2H7 scFv VHSER11 WTH WTCH2CH3, andtwo-fold serial dilutions were prepared. Serial two-fold dilutions ofpurified 2H7 scFv IgG MTH (SSC) WTCH2CH3 (SEQ ID NO:139) were preparedstarting at a concentration of 5 μg/ml. The culture supernatants andpurified fusion protein samples were incubated with (CD20+) CHO cellsfor one hour on ice. The cells were washed twice and then incubated with1:100 FITC-conjugated goat anti-human IgG (CalTag) for 40 minutes. Theunbound conjugate was then removed by washing the cells and flowcytometry analysis was performed using a Coulter Epics XL cell sorter.Results are presented in FIG. 34.

Example 17 Immunoblot Analysis of Anti-CD20 scFv Human IgG and IgAFusion Proteins

This Example describes immunoblot analysis of 2H7 scFv IgG and 2H7 scFvIgA fusion proteins that were immunoprecipitated from transfected cellculture supernants.

COS cells were transiently transfected with plasmids comprisingnucleotide sequences for 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:28);2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID NO:135); 2H7 scFv IgG MTH (SCS)WTCH2CH3 (SEQ ID NO:137); 2H7 scFv IgA H IgG WTCH2CH3 (SEQ ID NO:60);and scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO:58) essentially according tothe method described in Example 10. Cells were also transfected withvector only. After 48-72 hours at 37° C., cell culture supernatants wereharvested and combined with protein A-agarose beads (Repligen) for onehour at 4° C. The beads were centrifuged and washed several times inTNEN [20 mM Tris base, 100 mM NaCl, 1 mM EDTA, and 0.05% NP-40, pH 8.0).The immunoprecipitates were combined with 25 μl 2× NuPAGE® SDS SampleBuffer (Invitrogen Life Technologies) (non-reduced samples). Theproteins were fractionated on NuPAGE® 10% Bis-Tris gels (Invitrogen LifeTechnologies). After electrophoresis (approximately 1 hour), theproteins were transferred from the gel onto a Immobilon P polyvinylidenefluoride (PVDF) membrane (Millipore, Bedford, Mass.) using a semi-dryblotter (Ellard Instrumentation, Monroe, Wash.). The PVDF membrane wasblocked in PBS containing 5% nonfat milk and then probed withHRP-conjugated goat anti-human IgG (Fc specific) (CalTag). After washingthe immunoblot several times in PBS, the blot was developed using ECL(Amersham Biosciences). The results are shown in FIG. 35.

Example 18 Binding of Anti-CD20 scFv Human IgA Fusion Proteins toCD20+CHO Cells

This Example describes flow immunocytofluorimetry analysis of binding of2H7 scFv IgAH IgACH2CH3 (SEQ ID NO:62) and 2H7 scFv IgAH IgAT4 (SEQ IDNO:70) fusion proteins to (CD20+) CHO cells.

COS cells were transiently co-transfected as described in Example 10with plasmid DNA comprising a polynucleotide sequence (SEQ ID NO:61)encoding 2H7 scFv IgAH IgACH2CH3 polypeptide (SEQ ID NO:62) and with aseparate plasmid comprising a polynucleotide sequence (SEQ ID NO:65)encoding a human J chain polypeptide (SEQ ID NO:66). COS cells were alsotransfected with a polynucleotide sequence (SEQ ID NO:70) encoding a 2H7scFv IgA fusion protein that had a deletion of four amino acids at thecarboxy terminus of CH3 (2H7 scFv IgAH IgA-T4, SEQ ID NO:71). Thetransfections were performed as described in Example 10. Culturesupernatants from transfected COS cells were combined with (CD20+) CHOcells (see Example 1) and incubated for one hour on ice. The cells werewashed twice with PBS-2% FBS and then combined with FITC-conjugated goatanti-human IgA chain (CalTag) (1:100) for 40 minutes. The cells wereagain washed and then analyzed by flow cytometry using a Coulter EpicsXL cell sorter. FIG. 36 shows that co-transfection with J chain was notrequired for secretion of 2H7 scFv IgAH IgAT4, the 2H7 IgA fusionprotein with the truncated CH3 carboxy end (SEQ ID NO:71).

Example 19 Effector Function of Anti-CD20 scFv Human IgA Fusion Proteins

This Example illustrates ADCC activity of 2H7 IgG and IgA fusionproteins against cells that express CD20. BJAB cells were prelabeledwith ⁵¹Cr (100 μCi) (Amersham) for two hours at 37° C. Effector cellswere obtained from fresh, resting human whole blood, which was dilutedin an equal volume of Alsever's solution to prevent coagulation. 2H7scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO:58); 2H7 scFv IgG MTH (SCS)WTCH2CH3 (SEQ ID NO:137); 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ IDNO:28); and 2H7 scFv IgAH IgACH2CH3 (SEQ ID NO:62) fusion proteins werepurified from transiently transfected COS cell supernatants (100-200 ml)by protein A chromatography as described in Example 10. COS cellstransfected with the plasmid encoding 2H7 scFv IgAH IgACH2CH3 wereco-transfected with a plasmid encoding human J chain as described inExample 18. Two-fold serial dilutions of the purified 2H7 Ig fusionproteins starting at 5 μg/ml were added to the labeled BJAB cells (5×10⁴cells per well of 96 well tissue culture plate) in the presence of wholeblood (100 μl of whole blood diluted 1:1 in Alsever's solution, finaldilution 1:4) and incubated for five hours at 37° C. Culturesupernatants were harvested and analyzed as described in Example 11.Percent specific killing was calculated according to the followingequation: ((experiment release minus spontaneous release)/(maximumrelease minus spontaneous release))×100. The data are presented in FIG.37. Each data point represents the average of quadruplicate samples.

In a second ADCC assay, the number of labeled BJAB target cells was heldconstant in each sample, and whole blood was added at dilutions of 0.25,0.125, and 0.0625. Purified 2H7 IgG and IgA fusion proteins were addedat a concentration of 5 μg/ml. The BJAB cells, whole blood, and fusionproteins were incubated, the supernatants harvested, and the percentspecific killing was calculated as described above. Percent specifickilling for each of the 2H7 fusion proteins is presented in FIG. 38.

The ADCC activity of purified 2H7 scFv IgG MTH (SSS) WTCH2CH3 (5 μg/ml)and of purified 2H7 scFv IgAH IgACH2CH3 (5 μg/ml) was compared in thepresence of different effector cell populations. PBMC were isolated fromwhole blood as described in Examples 11 and 12. PBMC were combined withlabeled BJAB target cells (5×10⁴ per well of 96 well tissue cultureplate) at ratios of 50:1, 25:1, and 12.5:1. The assay was performed andthe data analyzed as described above. FIG. 39A shows that only the 2H7scFv IgG MTH (SSS) WTCH2CH3 fusion protein had ADCC activity when PBMCserved as the effector cells. FIG. 39B shows that both 2H7 scFv IgG MTH(SSS) WTCH2CH3 and 2H7 scFv IgAH IgACH2CH3 exhibit ADCC activity whenwhole blood was the source of effector cells (as illustrated in FIG.38).

Example 20 Expression Level of 2H7 scFv VH11ser IgG MTH (SSS) WTCH2CH3Fusion Protein

This Example compares the expression level of 2H7 scFv VH11Ser IgG MTH(SSS) WTCH2CH3 fusion protein (SEQ ID NO:370) with other 2H7 scFv IgGconstructs that do not contain the mutation in the variable heavy chaindomain. The mammalian expression vector pD18 comprising nucleotidesequences 2H7 scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO:370); 2H7 scFv IgGMTH (CSS) WTCH2CH3 (SEQ ID NO:372); 2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQID NO:137); 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:28); and 2H7 scFvVHSER11 IgG MTH (SSS) WTCH2CH3 (see Examples 1 and 13) were transientlytransfected into COS cells as described in Example 10. After 72 hours at37° C., culture supernatants were harvested, and 1 μl of eachsupernatant was combined with non-reducing sample buffer (see methoddescribed in Example 10). The culture supernatant samples and aliquotsof purified 2H7 scFv IgG MTH (SSS) WTCH2CH3 (40 ng, 20 ng, 10 ng/5 ng,and 2.5 ng) were fractionated on 10% Bis-Tris (MOPS) NuPAGE® gels(Invitrogen Life Technologies). Multimark® protein standards (InvitrogenLife Technologies) were also separated on the gel. The proteins weretransferred to a PDVF membrane and immunoblotted as described in Example17. The immunoblot is presented in FIG. 40. The amounts of the fusionproteins were quantified by densitometry analysis of the blots using theScionImage for Windows software and comparison with the standard curve.The 2H7 scFv IgG WTH (CCC) WTCH2CH3 construct produced approximately 12ng/ul or 12 micrograms/ml, the 2H7 scFv IgG MTH (CSS) WTCH2CH3 producedapproximately 10 ng/ul or 10 micrograms/ml, the 2H7 scFv IgG MTH (SCS)WTCH2CH3 construct produced approximately 1 ng/ul or 1 microgram/ml, andthe 2H7 scFv VHSER11 IgG MTH (SSS) WTCH2CH3 construct producedapproximately 30 ng/ml or 30 micrograms/ml. In particular claimedembodiments the instant invention, an amino acid sequence of 2H7 scFvVHSER11 IgG MTH (SSS) WTCH2CH3, or a polynucleotide sequence thatencodes 2H7 scFv VHSER11 IgG MTH (SSS) WTCH2CH3 may be optionallyexcluded from the instant invention. Similarly, an amino acid sequenceof 2H7 scFv VHSER11 IgG WTH (CCC) WTCH2CH3, or a polynucleotide sequencethat encodes 2H7 scFv VHSER11 IgG WTH (CCC) WTCH2CH3 may be optionallyexcluded from particular claimed embodiments of the instant invention.Additionally, an amino acid substitution of a leucine at position 11 toserine in the variable heavy chain domain, or polynucleotides thatencode an amino acid substitution of a leucine at position 11 to serinein the variable heavy chain domain, may be optionally excluded fromparticular claimed embodiments of the instant invention.

Example 21 Construction of a 2H7 scFv IgG Fusion Protein with a MutantCH3 Domain

Amino acid mutations were introduced into the CH3 domain of a 2H7 IgGfusion protein. The pD18 vector comprising 2H7 scFv IgG MTH (SSS)WTCH2CH3 (SEQ ID NO:135) was digested with BclI and XbaI to remove theMTH WTCH2CH3 fragment, which was then subcloned into pShuttle vector (BDBiosciences Clontech, Palo Alto, Calif.) that was double-digested withBclI and XbaI. Subcloning was performed in a kanamycin resistant vectorbecause the ampicillin resistance gene has an XmnI site, which isrequired for this cloning procedure. Five constructs were prepared withthe following substitutions: (1) a phenylalanine residue at position 405(numbering according to Kabat et al. supra) was substituted withtyrosine using the oligonucleotide CH3Y405; (2) the phenylalanineposition at 405 was substituted with an alanine residue using theoligonucleotide CH3A405; (3) the tyrosine residue at position 407 wassubstituted with an alanine using the oligonucleotide CH3A407; (4) bothwild type amino acids at positions 405 and 407 were substituted withtyrosine and alanine, respectively using the oligonucleotideCH3Y405A407; and (5) both wild type amino acids at positions 405 and 407were substituted with alanine using the oligonucleotide CH3A405A407. Theoligonucleotides were the 3′ primers for PCR amplification of a portionof the CH3 domain. The nucleotide sequences for each 3′ oligonucleotidewere as follows.

CH3Y405: (SEQ ID NO: 568) 5′-gtt gtt gaa gac gtt ccc ctg ctg cca cct gctctt gtc cac ggt gag ctt gct gta gag gta gaa gga gcc-3′ CH3A405: (SEQ IDNO: 569) 5′-gtt gtt gaa gac gtt ccc ctg ctg cca cct gct ctt gtc cac ggtgag ctt gct gta gag ggc gaa gga gcc-3′ CH3A407: (SEQ ID NO: 570) 5′-gttgtt gaa gac gtt ccc ctg ctg cca cct gct ctt gtc cac ggt gag ctt gct ggcgag gaa gaa gga gcc-3′ CH3Y405A407: (SEQ ID NO: 571) 5′-gtt gtt gaa gacgtt ccc ctg ctg cca cct gct ctt gtc cac ggt gag ctt gct ggc gag gta gaagga gcc-3′ CH3A405A407: (SEQ ID NO: 572) 5′-gtt gtt gaa gac gtt ccc ctgctg cca cct gct ctt gtc cac ggt gag ctt gct ggc gag ggc gaa gga gcc-3′

The template was the mutant hinge MHWTCH2CH3 human IgG1. The 5′ PCRoligonucleotide primer was huIgGMHWC. The amplified products were TOPO®cloned and sequenced as described in Examples 1 and 10. DNA from theclones with the correct sequence was digested with BclI and XmnI andtransferred to pShuttle containing the MTH WTCH2CH3 sequence, which wasalso digested with the same restriction enzymes. The mutated IgGsequences were then removed by digestion with BclI and XbaI and insertedinto a pD18 vector containing 2H7 scFv that was also digested with BclIand XbaI. The polynucleotide sequences for mutated the CH3 domains,MTCH3 Y405, MTCH3 A405, MTCH3 A407, MTCH3 Y405A407, and MTCH3 A405A407are shown in SEQ ID NOs:143, 145, 147 and 149, respectively, and thepolypeptide sequences for each are shown in SEQ ID NOs:144, 146, 148 and150, respectively. The polynucleotide sequences for the 2H7 scFv MTHWTCH2 MTCH3 Y405, 2H7 scFv MTH WTCH2 MTCH3 A405, scFv MTH WTCH2 MTCH3A407, scFv MTH WTCH2 MTCH3 Y405A407, and scFv MTH WTCH2 MTCH3 A405A407,respectively, and the deduced amino acid sequences are shown in SEQ IDNOs:154, 156, 158, 160 and 162, and the deduced nucleotide sequences areshown in SEQ ID NOs: 153, 155, 157, 159, 161, respectively,respectively.

Example 22 Construction of 2H7 scFv IgG Fusion Proteins with HingeMutations

A 2H7 scFv IgG fusion protein was constructed with the third cysteineresidue in the IgG1 hinge region substituted with a serine residue. Thetemplate for introduction of the mutations was a polynucleotide encoding2H7 scFv WTH WTCH2CH3 (SEQ ID NO:28). The oligonucleotide introducingthe mutations was a 5′ PCR primer oligonucleotide HIgGMHcys3 having thesequence 5′-gtt gtt gat cag gag ccc aaa tct tgt gac aaa act cac aca tgtcca ccg tcc cca gca cct-3′ (SEQ ID NO:573). The oligonucleotideintroducing the mutation into the hinge region was combined withtemplate and a 3′ oligonucleotide containing an XbaI site (underlinedand italicized) (5′-gtt gtt tct aga tca ttt acc cgg aga cag gga gag getctt ctg cgt gta g-3′ (SEQ ID NO:574)) to amplify the mutant hinge-wildtype (WT)-CH2-CH3 sequences by PCR. The IgG MTH CCS mutant sequence wasamplified for 30 cycles with a denaturation profile of 94° C., annealingat 50° C. for 30 seconds, and extension at 72° C. for 30 seconds. Theamplified polynucleotides were inserted into the TOPO® cloning vector(Invitrogen Life Technologies) and then were sequenced as described inExample 1 to confirm the presence of the mutation. pD18 vectorcontaining 2H7 scFv was digested to remove the constant region sequencesessentially as described in Example 10. The mutant hinge-wild typeCH2-CH3 regions were inserted in frame into the digested vector DNA toobtain vectors comprising 2H7 scFv MTH (CCS) WTCH2CH3 encoding DNA (SEQID NO:167). The deduced polypeptide sequence is shown in SEQ ID NO:168.

Example 23 Construction of Anti-CD20 IgE Fusion Proteins

A binding domain is fused to IgE constant region sequences such that theexpressed polypeptide is capable of inducing an allergic responsemechanism. The single chain Fv nucleotide sequence of 40.2.220 (SEQ IDNO:35), an anti-CD40 antibody, is fused to IgE CH2-CH3-CH4 according tomethods described for other scFv immunoglobulin constant regionconstructs (see Examples 1, 5, 10, and 13). To PCR amplify the IgECH2-CH3-CH4 domains, a 5′ oligonucleotide primer, hIgE5Bcl, having thesequence 5′-gtt gtt gat cac gtc tgc tcc agg gac ttc acc cc-3′ (SEQ IDNO:575), and a 3′ oligonucleotide primer, hIgE3stop, having the sequence5′-gtt gtt tct aga tta act ttt acc ggg att tac aga cac cgc tcg ctg g-3′(SEQ ID NO:576) are used.

The retroviral transfection system for ectopic surface expression ofgenetically engineered cell surface receptors composed of scFvs thatbind costimulatory receptors described in Example 12 is used toconstruct a 40.2.220 scFv IgE-CD80 fusion protein. The 40.2.220 scFv IgEfusion polynucleotide sequence is fused in frame to sequences encodingthe transmembrane domain and cytoplasmic tail of human CD80 (SEQ IDNO:30), such that when the fusion protein is expressed in thetransfected cell, CD80 provided an anchor for surface expression of thescFv Ig fusion protein. cDNA encoding the anti-CD40 scFv-IgE-CD80 fusionproteins is inserted into the retroviral vector pLNCX (BD BiosciencesClontech) according to standard molecular biology procedures and vendorinstructions. The 40.2.220 scFv-Ig-CD80 cDNA is inserted between the5′LTR-neomycin resistance gene-CMV promoter sequences and the 3′LTRsequence. The retroviral constructs are transfected into a carcinomacell line, and transfected cells are screened to select clones that areexpressing the 40.2.220 scFv-Ig-CD80 fusion protein on the cell surface.

Example 24 Construction of IgA-T4 Mutants that are Expressed on the CellSurface

The retroviral transfection system for ectopic surface expression ofgenetically engineered cell surface receptors composed of scFvs thatbind costimulatory receptors described in Example 12 is used toconstruct a 2H7 scFv IgA hinge IgA-T4-CD80 fusion protein. The 2H7 scFvIgAH IgA-T4 fusion polynucleotide sequence (SEQ ID NO:70) is fused inframe to sequences encoding the transmembrane domain and cytoplasmictail of human CD80 (SEQ ID NO:29), such that when the fusion protein isexpressed in the transfected cell, CD80 provided an anchor for surfaceexpression of the scFv Ig fusion protein. cDNA encoding the 2H7 scFvIgAH IgA-T4-CD80 fusion protein is inserted into the retroviral vectorpLNCX (BD Biosciences Clontech) according to standard molecular biologyprocedures and vendor instructions. The 2H7 scFv IgAH IgA-T4-CD80 cDNAis inserted between the 5′LTR-neomycin resistance gene-CMV promotersequences and the 3′LTR sequence. The retroviral construct istransfected into Reh, an acute lymphocytic leukemia cell line (ATCCCRL-8286). Transfected cells are screened to select clones that areexpressing 2H7 scFv-Ig fusion proteins on the cell surface.

Example 25 Characterization of an Anti-4-1BB scFv Ig-CD80 Fusion ProteinExpressed on the Cell Surface of Tumor Cells and Growth of the TumorCells In Vivo

This Example describes construction of an anti-murine 4-1BB (CD137) scFvfusion protein that has an IgG wild type hinge and CH2 and CH3 domainsthat is fused to the CD80 transmembrane and cytoplasmic domains. TheExample also illustrates the effect of the cell surface expression ofthe anti-4-1BB scFv IgG CD80 polypeptide when the transfected tumorcells are transplanted into mice.

The heavy and light chain variable regions of a rat anti-4-1BB (CD137)monoclonal antibody (1D8) were cloned, and a single chain Fv constructwas prepared essentially as described in Example 1. The heavy chain andlight chain variable regions of each antibody were cloned according tostandard methods for cloning immunoglobulin genes and as described inExample 1. Aingle chain Fv construct was prepared as described inExample 1 by inserting a nucleotide sequence encoding a (gly₄ser)₃ (SEQID NO:529) peptide linker between the VL region nucleotide sequence of1D8 (SEQ ID NO:102) and the VH region nucleotide sequence of 1D8 (SEQ IDNO:100). The polypeptide sequence for 1D8 VL is shown in SEQ ID NO:103,and the polypeptide sequence for the VH domain is shown in SEQ IDNO:101. The scFv polynucleotide (SEQ ID NO:104) was then fused to humanIgG1 wild-type hinge-CH2-CH3 domains according to the methods describedin Example 1. The scFv IgG1 fusion polynucleotide sequence was thenfused in frame to sequences encoding the transmembrane domain andcytoplasmic tail of human CD80 (SEQ ID NO:29) essentially as describedin Example 12, such that when the fusion protein was expressed in thetransfected cell, CD80 provided an anchor for surface expression of thescFv Ig fusion protein. cDNA encoding the scFv-IgG-CD80 fusion protein(SEQ ID NO:110) was inserted into the retroviral vector pLNCX (BDBiosciences Clontech) according to standard molecular biology proceduresand vendor instructions. The scFv-Ig-CD80 cDNA was inserted between the5′LTR-neomycin resistance gene-CMV promoter sequences and the 3′LTRsequence.

The retroviral constructs were transfected into the metastatic M2 cloneof K1735, a melanoma cell line, provided by Dr. I. Hellstrom, PNRI,Seattle, Wash. Transfected cells were screened to select clones thatwere expressing scFv-Ig fusion proteins on the cell surface. Todemonstrate that the 1D8 scFv IgG-CD80 construct was expressed on thecell surface of the tumor cells, the transfected cells were analyzed byflow immunocytofluorimetry. Transfected cells (K1735-1D8) were incubatedfor one hour on ice in phycoerythrin-conjugated F(ab′)₂ goat anti-humanIgG. The unbound conjugate was then removed by washing the cells andflow cytometry analysis was performed using a Coulter Epics XL cellsorter. Results are presented in FIG. 41A.

The growth of K1735-1D8 transfected cells was examined in vivo. K1735-WTcells grew progressively when transplanted subcutaneously (s.c.) innaïve C3H mice. Although the same dose of K1735-1D8 cells initiallyformed tumors of an approximately 30 mm² surface area, the tumorsstarted to regress around day 7 and had disappeared by day 20 as shownin FIG. 41B. Tumor cells that were transfected with a similarlyconstructed vector encoding a non-binding scFv, a human anti-CD28 scFvconstruct, grew as well as tumor cells that had not been transfected.The presence of a foreign protein, that is, human IgG1 constant domainsor rat variable regions, did not make transfected K1735-WT cellsimmunogenic; the growth of the K1735-1D8 cells in C3H mice was identicalto that of K1735-WT cells (untransfected).

To investigate the roles of CD4⁺ and CD8⁺ T lymphocytes and NK cells inthe regression of K1735-1D8 tumors, naïve mice were injectedintraperitoneally (i.p.) with monoclonal antibodies (monoclonalantibodies, typically 50 μg in a volume 0.1 ml) to remove CD8⁺, CD4⁺ orboth CD4⁺ and CD8⁺ T cells, or were injected with anti-asialo-GM1 rabbitantibodies to remove NK cells. Twelve days later, when flow cytometryanalysis of spleen cells from identically treated mice showed that thetargeted T cell populations were depleted, K1735-1D8 cells weretransplanted s.c to each T cell-depleted group. K1735-1D8 had similargrowth kinetics in mice that had been injected with the anti-CD8 MAb orcontrol rat IgG while removal of CD4′ T cells resulted in the growth ofK1735-1D8 with the same kinetics as K1735-WT. This failure to inhibittumor growth after CD4+ T cell removal was observed regardless of thepresence or absence of CD8+ T cells. K1735-1D8 grew in all NK-depletedmice, although more slowly than in the CD4-depleted group. The resultsare presented in FIG. 41C.

Example 26 Therapeutic Effect of Tumor Cells Expressing Anti-4-1BB scFvIgG-CD80 Fusion Protein

This Example examines the ability of K1735-1D8 transfected tumor cellsexpressing an anti-CD137 scFv on the cell surface to generate asufficient immune response in mice to mediate rejection of established,untransfected wild type tumors. C3H mice were transplanted with K1735-WTtumors (2×10⁶ cells/animal) and grown for approximately six days.Experiments were performed using mice with established K1735-WT tumorsof 30 mm² surface area. Mice were vaccinated by s.c. injection ofK1735-1D8 or irradiated K1735-WT cells on the contralateral side.Identical injections were repeated at the time points indicated in FIG.42. One group of animals was given four weekly injections of K1735-1D8cells. According to the same schedule, another group was givenirradiated (12,000 rads) K1735-WT cells, and a third group was injectedwith PBS. The data are plotted in FIG. 42. The WT tumors grewprogressively in all control mice and in all mice that receivedirradiated K1735-WT cells. In contrast, the tumors regressed in 4 of the5 mice treated by immunization with K1735-1D8. The animals remainedtumor-free and without signs of toxicity when the experiment wasterminated 3 months later. In the fifth mouse, the tumor noduledecreased in size as long as the mouse received K1735-1D8 cells, but thetumor grew back after therapy was terminated.

In another experiment with 5 mice/group, mice were injectedintravenously (i.v.) with 3×10⁵ K1735-WT cells to initiate lungmetastases. Three days later, K1735-1D8 cells were transplanted s.c.This procedure was repeated once weekly for a month; control mice wereinjected with PBS. The experiment was terminated when one mouse in thecontrol group died, 37 days after receiving the K1735-WT cells. At thattime, lungs of the control mice each had >500 metastatic foci. Incontrast, less than 10 metastatic foci were present in the lungs of theimmunized mice.

In a third experiment, mixtures of K1735-WT cells and K1735-1D8 cellswere injected into immunocompetent syngeneic C3H mice. Mice wereinjected subcutaneously with K7135-WT cells alone or with a mixture of2×10⁶ K1735-WT cells and 2×10⁵ K1735-1D8 cells. Tumor growth wasmonitored at 5-day intervals.

Example 27 Expression of Anti-4-1BB scFv IgG-CD80 Fusion Protein on theCell Surface of Sarcoma Cells

This Example demonstrates expression of an anti-CD137 scFv on the cellsurface of a second type of tumor cell by transfecting a murine sarcomacell line with an anti-CD137 scFv IgG-CD80 construct.

The 1D8 scFv IgG WTH WTCH2CH3-CD80 polynucleotide (SEQ ID NO:110) wastransferred from the pLNCX vector into pcDNA3-hygro vector usingrestriction enzyme digestion and ligation steps according to standardmolecular biology methods. The constuct was cut with HindIII+Cla1 andthesFv fragment was filled in with Klenow (Roche) and the blunt-endedfragment was ligated into EcoR5 site of pcDNA3. Ag104 murine sarcomatumor cells were transfected with the pcDNA3-hygro vector containing the1D8 scFv IgG CD80 fusion protein. Hygromycin-resistant clones werescreened by flow cytometry using a FITC anti-human IgG antibody todetect expression of the transgene. Only approximately 15% of theresistant clones had detectable fusion protein initially. Positive cellsidentified by flow cytometry were repeatedly panned on flasks coatedwith immobilized anti-human IgG (10 μg/ml) according to standardmethods. Panning was performed by incubating cells on the coated platesfor 30 min at 37 C; the plates were then washed 2-3× in versene or PBS.After each round, cells were tested for IgG expression by FACS. Thehistogram in FIG. 44 shows the staining pattern after four rounds ofpanning against anti-human IgG (black). Untransfected cells were stainedand are indicated in gray. All of the cells in the population werepositive.

Example 28 Construction and Characterization of a Bispecific scFv IgFusion Protein and scFv Ig Fusion Proteins with a Mutation in the IgG1CH2 Domain

An anti-CD20 (2H7) scFv IgG fusion protein was constructed that had amutant hinge (MT (SSS)) and a mutant CH2 domain in which the proline atresidue (position number 238 according to Ward et al., supra) wassubstituted with a serine. The 2H7 scFv IgG MTH (SSS) MTCH2WTCH3encoding polynucleotide (SEQ ID NO:130) was constructed essentiallyaccording to methods described in Examples 1, 5, and 13. The IgG mutanthinge-mutant CH2-wild type CH3 domains were also fused to an anti-CD20(2H7)-anti-CD40 (40.2.220) bispecific scFv. The anti-CD20-anti-CD40 scFvIgG MTH (SSS) MTCH2WTCH3 encoding polynucleotide sequence is shown inSEQ ID NO:114 and the encoded polypeptide is shown in SEQ ID NO:115.

COS cells were transiently transfected with vectors comprising thepolynucleotide sequences encoding 2H7 scFv IgG MTH (SSS) MTCH2WTCH3 (SEQID NO:131); anti-CD20-anti-CD40 scFv IgG MTH (SSS) MTCH2WTCH3 (SEQ IDNO:114); 2H7 scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO:58); and 2H7 scFvIgAH IgG WTCH2CH3 (SEQ ID NO:60) as described in Example 10. Culturesupernatants were collected and the fusion proteins were purified byprotein A chromatography (see Example 10). The purified polypeptideswere fractionated by SDS-PAGE according to the method described inExample 10. Rituximab (anti-CD20 monoclonal antibody), and Bio-Radprestained molecular weight standards (Bio-Rad, Hercules, Calif.), andMultimark® molecular weight standards (Invitrogen Life Technologies werealso applied to the gel. The results are presented in FIG. 45.

The 2H7 scFv Ig fusion protein that contains a mutation in the CH2domain was compared to fusion proteins that have the wild type CH2domain in an ADCC assay. The assays were performed essentially asdescribed in Examples 11 and 19. Fresh resting PBMC (effector cells)were added to ⁵¹Cr-labeled BJAB cells (target cells) at the ratiosindicated in FIG. 46. Purified 2H7 scFv IgG MTH (SSS) MTCH2WTCH3, 2H7scFv IgG MTH (SSS) WTCH2CH3, 2H7 scFv IgAH IgG WTCH2CH3, and Rituximab,each at 10 μg/ml were added to the effector/target cell mixtures andincubated for five hours at 37° C. Supernatants were harvested and theamount of chromium released was determined as described in Examples 11and 19. Percent specific killing by each fusion protein is presented inFIG. 46.

Example 29 Tumor Cell Surface Expression of an Anti-Human CD3 scFv IgGFusion Protein

An anti-human CD3 scFv Ig CD80 fusion protein was prepared essentiallyas described in Examples 1 and 12. The fusion protein comprised ananti-human CD3 scFv fused to wild type IgG1 hinge (SEQ ID NO:12) andwild type CH2 (SEQ ID NO:13) and CH3 (SEQ ID NO:15) domains, fused toCD80 transmembrane and cytoplasmic domains (SEQ ID NO:29) to enable cellsurface expression of the anti-CD3 scFv. The anti-human CD3 scFv IgG WTHWTCH2CH3-CD80 polynucleotide (SEQ ID NO:110) encoding the polypeptide(SEQ ID NO:111) was transfected in Reh cells and into T51 cells(lymphoblastoid cell line). Expression of the anti-human CD3 scFv IgGfusion protein was detected by flow cytometry using FITC conjugated goatanti-human IgG (see methods in Examples 4, 10, 16, 18). FIG. 47Aillustrates expression of the anti-human CD3 fusion protein on the cellsurface of Reh cells, and FIG. 47B shows expression of the fusionprotein on T41 cells.

ADCC assays were performed with the transfected Reh and T51 cells todetermine if expression of the scFv-Ig polypeptides on the cell surfaceaugmented effector cell function. Untransfected and transfected Rehcells and untransfected and transfected T51 cells were pre-labeled with⁵¹Cr (100 μCi) (Amersham) for two hours at 37° C. Human PBMC served aseffector cells and were added to the target cells (5×10⁴ cells per wellof 96 well plate) at ratios of 20:1, 10:1, 5:1, and 2.5:1. After fourhours at 37° C., culture supernatants were harvested and analyzed asdescribed in Examples 11 and 12. Percent specific killing was calculatedas described in Example 12. The results are presented in FIG. 48.

Example 30 Induction of Cytokine Expression in Tumor Cells ExpressingAnti-CD28 scFv on the Cell Surface

This Example describes the effect of cell surface expressed scFv oncytokine mRNA induction in stimulated lymphocytes co-cultured with tumorcells transfected with an anti-human CD28 scFv IgG-CD80 fusion protein.

Real time PCR analysis was performed on RNA samples from human PBMCstimulated with Reh, Reh-anti-CD28 (2e12) (see Example 12 forconstruction of 2e12 scFv IgG WTH WHTCH3CH2-CD80 and transfection of Rehcells), and Reh-CD80 (see Example 14) in order to measure the effects ofthe surface expressed scFv on cytokine production by the PBMC effectorcells. For the real-time PCR assay, SYBR Green (QIAGEN) (Morrison etal., Biotechniques 24:954-8, 960, 962 (1998)) was used and measured byan ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, FosterCity, Calif.) that measures the formation of PCR product after eachamplification cycle. Cells were harvested from cultures and total RNAprepared using QIAGEN RNA kits, including a QIA shredder columnpurification system to homogenize cell lysates, and RNeasy® mini-columnsfor purification of RNA. cDNA was reverse transcribed using equalamounts of RNA from each cell type and Superscript II ReverseTranscriptase (Life Technologies). SYBR Green real-time PCR analysis wasthen performed using the prepared cDNA as template and primer pairsspecific for cytokine gene products. The average length of the PCRproducts that were amplified ranged from 150-250 base pairs. The cDNAlevels for many activation response molecules including IFNγ, TNFα,GM-CSF, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, ICOSL, CD80 andCD86 were assayed. Control reference cDNAs for constitutively expressedgenes, including GAPDH, β-actin, and CD3ε were measured in each assay.The most significant induction of specific mRNA was observed for IFN-γ,and more modest induction was observed for CTLA-4 and ICOS.

Example 31 Cloning of an Anti-Human 4-1BB Antibody and Construction ofan Anti-Human 4-1BB scFv Ig Fusion Protein

A hybridoma cell line expressing a mouse anti-human monoclonal antibody(designated 5B9) was obtained from Dr. Robert Mittler, Emory UniversityVaccine Center, Atlanta, Ga. The variable heavy and light chain regionswere cloned according to known methods for cloning of immunoglobulingenes and as described herein. Cells were grown in IMDM/15% FBS(Invitrogen Life Technologies) media for several days. Cells inlogarithmic growth were harvested from cultures and total RNA preparedusing QIAGEN RNA kits, including a QIA shredder column purificationsystem to homogenize cell lysates, and RNeasy® mini-columns forpurification of RNA according to manufacturer's instructions. cDNA wasreverse transcribed using random hexamer primers and Superscript IIReverse Transcriptase (Invitrogen Life Technologies).

cDNA was anchor-tailed using terminal transferase and dGTP. PCR was thenperformed using an anchor-tail complementary primer and a primer thatannealed specifically to the antisense strand of the constant region ofeither mouse Ck (for amplifcation of VL) or the appropriate isotypemouse CH1 (for amplification of VH). The amplified variable regionfragments were TOPO® cloned (Invitrogen Life Technologies), and cloneswith inserts of the correct size were then sequenced. Consensus sequencefor each variable domain was determined from sequence of at least fourindependent clones. The 5B9 VL and VH polynucleotide sequences are shownin SEQ ID NOs:120 and 116, respectively, and the deduced amino acidsequences are shown in SEQ ID NOs:121 and 118. The scFv was constructedby a sewing PCR method using overlapping primers containing a synthetic(Gly₄Ser)₃ (SEQ ID NO:529) linker domain inserted between the light andheavy chain variable regions (see Example 1). The 5B9 scFv polypeptide(SEQ ID NO:123) is encoded by the polynucleotide sequence comprising SEQID NO:122.

5B9 scFv polynucleotide sequence was fused in frame to thepolynucleotide sequence encoding the human IgG1 mutant hinge and wildtype CH2 and CH3 (MTH (SSS) WTCH2CH3, according to methods described inExamples 5, 10, and 13. COS cells were transiently transfected with avector comprising the 5B9 scFv IgG MTH (SSS) WTCH2CH3 polynucleotidesequence (SEQ ID NO:132). Supernatant was collected and binding of the5B9 scFv IgG MTH (SSS) WTCH2CH3 polypeptide (SEQ ID NO:133) was measuredby flow immunocytofluorimetry essentially as described in Examples 4,10, 16, and 18. Culture supernatant from the 5B9 hybridoma cell line wasalso included in the binding assay. Fresh human PBMC were incubated inthe presence of immobilized anti-CD3 for four days prior to the bindingexperiment to induce expression of CD137 on the surface of activated Tcells. Stimulated PBMC were washed and incubated with COS or hybridomaculture supernatant containing the 5B9 scFv IgG fusion protein or 5B9murine monoclonal antibody, respectively, for 1 hour on ice. Binding of5B9 scFv IgG fusion protein or 5B9 murine monoclonal antibody wasdetected with FITC conjugated anti-human IgG or anti-mouse IgG,respectively. The results are presented in FIG. 49.

Example 32 Construction of 2H7 scFv IgG Fusion Proteins with HingeMutations

2H7 scFv IgG fusion proteins are constructed with the first cysteineresidue and the second cystein in the IgG1 hinge region substituted witha serine residue to provide MTH (SCC) and MTH (CSC). The template forintroduction of the mutations is a polynucleotide encoding 2H7 scFv WTHWTCH2CH3. The oligonucleotide introducing the mutations are 5′ PCRprimer oligonucleotides HIgGMHcys1 (SEQ ID NO:140) and HIgGMHcys2 (SEQID NO:141). The constructs are prepared as described previously. Theencoding polynucleotides of the mutants are presented in SEQ ID NOs:163and 165) and the polypeptide sequences are provided in SEQ ID NO:164 and166).

Example 33 Construction of 2H7 V_(H)L11S scFv (SSS-S) H WCH2 WCH3

A change from leucine to serine at position 11 in the heavy chainvariable region (numbering according to Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) ed. Bethesda, Md.: PublicHealth Service, National Institutes of Health (1991)) was introducedinto the 2H7 scFv MTH (SSS) WTCH2CH3 fusion protein (SEQ ID NO:58). Thewild type leucine residue was substituted with serine by site-directedmutagenesis using the oligonucleotide Vhser11: 5′-gga ggt ggg agc tctcag gct tat cta cag cag tct ggg gct gag tcg gtg agg cc-3′ (SEQ IDNO:577). The 3′-primer for PCR was huIgG1-3′ having the sequence 5′-gtctct aga cta tca ttt acc cgg aga cag-3′ (SEQ ID NO:578) (XbaI siteunderlined and italicized). After PCR amplification, the fragments wereinserted into the TOPO® cloning vector and sequenced to confirm thepresence of the VH11 leucine to serine mutation. The 2H7 scFv-IgG(SSS-S) H WCH2 WCH3 encoding DNA was shuttled into the PSL1180 cloningvector (Pharmacia Biotech, Inc., Piscataway, N.J.). The constructPSL1180-2H7 scFv-IgG (SSS-S) H WCH2 WCH3 was digested with Sac and XbaIto remove the wild type VH domain and the connecting region and CH2 andCH3 domains. The PCR product comprising the VH11 mutant was digestedwith Sac and XbaI and then inserted into the digested PSL1180 constructaccording to standard molecular biology procedures. The construct wasthen digested with Hind III and XbaI, and inserted into the mammalianexpression vector pD18 (see methods described in Example 1 and Example10). The mutant is designated 2H7 scFv VH L11S IgG (SSS-S) H WCH2 WCH3.The polynucleotide sequence is provided in SEQ ID NO:369, and theencoded polypeptide sequence is provided in SEQ ID NO:370.

Example 34 Expression and of 2H7 scFv VH L11S (SSS-S) H WCH2 WCH3 inStable CHO Lines

CHO DG44 cells were transfected by electroporation with approximately150 micrograms of linearized expression plasmid encoding the 2H7 VH L11S scFv (SSS-S) H WCH2 WCH3. Cultures were plated in selection mediacontaining 100 nM methotrexate, in 96 well, flat bottom tissue cultureplates at various numbers of cells/well, ranging from 125 to 2000.Methotrexate resistant clones were selected and culture supernatantswere screened for the highest expressors of the fusion protein using aCD20CHO binding assay similar to that described for FIG. 1. Clones wereamplified after the initial selection in gradually increasing doses ofmethotrexate. Cells were passaged for two passages in the higherconcentration prior to adjusting the concentration to the next higherdose. Clones were amplified to a final concentration of 1 micromolarmethotrexate.

FIG. 50B illustrates the production levels of 2H7 VH L11S scFv (SSS-S) HWCH2 WCH3. Spent supernatants from amplified CHO cells expressing thismolecule and growing in stationary T25 flasks were tested forquantitative binding to CD20 CHO cells by flow cytometry. The activitywas converted to protein concentration by generation of a standard curveusing the same molecule purified from supernatants with Protein Aaffinity chromatography (FIG. 50A). The concentration of the purifiedprotein was determined by A280 using an extinction coefficient providedby the amino acid composition of the recombinant protein (Vector NTI).Although levels of production varied between clones tested, multipleclones produced over 1 mg/ml. This level of protein expression is over10-fold higher than the identical molecule except for the amino acidchange in V_(H).

FIG. 51 illustrates the production levels of 2H7 VH L11S scFv (SSS-S) HWCH2 WCH3 by semi-quantitative analysis on SDS-PAGE. Ten microliters ofspent supernatant from amplified CHO cells expressing this molecule andgrowing in stationary T25 flasks were mixed with 10 microliters 2×non-reducing SDS sample buffer, run on SDS-PAGE gels, and stained withcoomassie blue.

Example 35 Construction and Binding Capacity of G28-1 scFv Ig Constructs

Contrstruction of the G28-1 (anti-CD37) scFv was performed using totalRNA isolated from the G28-1 hybridoma using Trizol (Invitrogen) reagentaccording to manufacturer's instructions. cDNA was prepared using randomprimers and the protocol described previously for 2H7 cloning inExample 1. The variable domains of the scFv was cloned using one of twomethods: the first method used a family of degenerate 5′oligonucleotides specific for each V region gene family and a single 3′primer specific for the constant region of either the light or heavychain using methods and primers described in (Ig-Prime Kit MouseIg-Primer Set, Novagen). The second approach used the anchor-tailingmethods and primers described in (Gilliland L K et al, Tissue Antigens47: 1-20 (1995). In either case, PCR amplified products were cloned intothe TOPO cloning vector (Invitrogen). The clones were digested withEcoRI and screened for inserts of the proper size. Positive clones weresequenced as previously described in Example 1.

Specific primers were then designed for each V region, one with theleader sequence and one without. Primers were also designed to includedesired linkers and/or restriction sites at the primer ends. PCRreactions were performed on the TOPO cloned DNA using a 25 cycle programwith the following profile: 94 C, 30 sec; 55 C, 30 sec; 72 C, 30 sec,followed by a final extension at 72 C for 8 minutes. PCR products weregel purified and fragments recovered using a QIAQUICK gel extraction kit(QIAGEN, Valencia, Calif.). Fragments were diluted 1:50 and 1 microliterused for SEWING PCR reactions according to the methods described inExample 1. The following oligonucleotides were used for the secondaryPCR reactions of the V_(L) domain for the G28-1 scFv:

5′ primers with SalI site without leader: (SEQ ID NO: 579)5′-GTTGTTGTCGACATCCAGATGACTCAGTC TCCA-3′ 5′ primer with HindIII site andleader sequence: (SEQ ID NO: 580) 5′-GTCAAGCTTGCCGCCATGGTATCCACAGCTCAGTTCCTTGG-3′ 3′ primer: (SEQ ID NO: 581)5′-GCCACCCGACCCACCACCGCCCGAGCCACCGCCACCTTTGATCTCCA GTTCGGTG CC-3′

The primers used for the V_(H) domain are shown below:

5′ sense primer: (SEQ ID NO: 582)TCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCGTCAGCGGTCCAGCTGCA GCAGTCTGGA-3′3′ antisense primer with BclI site: (SEQ ID NO: 583)5′-TCAGTGCTGATCAGAAGAGACGGTGACTGAGGTTCCTTG-3′.

A change from leucine to serine at position 11 in the heavy chainvariable region (Kabat numbering) was introduced in into the G28-1 scFvby site-directed mutagenesis. The wild type form of the G28-1 scFv wasinitially constructed by sewing/overlap PCR to insert a (gly4ser)3 (SEQID NO:529) linker between the VL and VH domains as described above.However, no Sac I site was introduced as a part of this fusion of thevariable domains, so alternative, nearby restriction sites (HaeII andPvuII) near leucine 11 were used to synthesize the VL+mutated VH domain.Primers were designed to contain one of these sites and the DNA sequenceincluding the L to S change, followed by 12 wild type base pairs.Several attempts at this strategy failed, so an alternative strategyusing the Genetailor (Invitrogen) method of site directed mutagenesiswas used to introduce the desired mutation. The mutagenesis was carriedout according to manufacturer's instructions. Briefly the procedureinvolves methylation of the plasmid DNA with DNA methylase,amplification of the DNA in a mutagenesis reaction with two overlappingprimers, one of which contains the target muations, trasformation of theplasmid into wild type E. coli which digests all methylated DNA andleaves only the unmethylated, mutated amplification product. Bothprimers are approximately 30 nucleotides in length (not including themutation site on the mutagenic primer, with an overlapping region at the5′ ends of 15-20 nucleotides, for efficient end joining of themutagenesis product. The template for the mutagenesis reaction was 100ng of a plasmid containing the wild type G28-1 scFvIg construct, and theprimers used for the G28-1 VH mutagenesis are as follows:

Forward primer: (SEQ ID NO: 584) 5′-GCAGCAGTCTGGACGTGAGTCGGAAAAGCCTG-3′Reverse Primer: (SEQ ID NO: 585) 5′-CTCAGGTCCAGACTGCTGCAGCTGGACCGC-3′

PCR reactions were performed using the 15 ng methylated template, theprimers above, and the usual reaction components as previouslydescribed. A 20 cycle program with the following profile was used foramplification: 94 C, 30 sec; 55 C, 30 sec; 68 C, 8 min, followed by afinal 68 C extension step for 10 minutes. PCR products were transformedinto wild type bacteria, and colonies screened by sequencing. Cloneswith only the desired mutation were isolated and plasmid prepared aspreviously described Example 33. The mutant is designated G28-1 scFv VHL11S (SSS-S)H WCH2 WCH3. The polynucleotide sequence is provided in SEQID NO:323, and the encoded polypeptide sequence is provided in SEQ IDNO:324.

The expression level of G28-1 fusion proteins was confirmed usingImmunblot analysis according to the methods described in Example 17.FIG. 53 illustrates a large increase in protein expression in the VHL11S mutant G28-1 fusion proteins compared to the G28-1 fusion proteinwithout the mutation.

The G28-1 scFv Ig fusion proteins were transiently transfected andexpressed in COS cells according to methods described in Example 10.FIG. 52 illustrates the capacity of the G28-1 scFv Ig fusion proteinsfrom the COS supernatants to bind CD37. Ramos and BJAB cells bothexpress human CD37, and were therefore used to screen the G28-1supernatants for functional activity. Binding of G28-1 scFv (SSS-S) HWCH2 WCH3 and G28-1 scFv VH L11S (SSS-S) H WCH2 WCH3 to CD37+ Ramoscells was measured by flow cytometry according to methods described inExample 2. Each point on the graph represents the mean of five replicatetransfections. The graph illustrates that the G28-1 scFv VH L11S (SSS-S)H WCH2 WCH3 is able to bind CD37+ Ramos cells.

Addition constructs were made with different connecting regions. ThepD18 G28-1 scFv VHL11S (SSS-S) H WCH2 WCH3 vector was digested with BclIand XbaI to remove the connecting region, CH2 and CH3. Theses werereplaced with each different connecting region, CH2 and CH3 according tothe methods described in Example 13. The new constructs were designated:G28-1 scFv VHL11S (CSS-S) H WH2 WH3 (SEQ ID NO:325), G28-1 scFv VHL11S(CSC-S) H WH2 WH3 (SEQ ID NO:327), G28-1 scFv VHL11S (SSC-P) H WH2 WH3(SEQ ID NO:329), G28-1 scFv VHL11S (SCS-S) H WH2 WH3 (SEQ ID NO:373),G28-1 scFv VHL11S (CCS-P) H WH2 WH3 (SEQ ID NO:375), and G28-1 scFvVHL11S (SCC-P) H WH2 WH3 (SEQ ID NO:377).

The G28-1 scFv was also attached to an IgA connecting region, CH2, CH3and an IgE CH2, CH3, CH4. The pD18 G28-1 scFv VHL11S (SSS-S) H WCH2 WCH3plasmid was digested using methods above to remove the connecting regionCH2, and CH3. The IgA regions were inserted using methods described inExample 13. The construct was designated G28-1 scFv VHL11S IgAH IgACH2T4CH3 (SEQ ID NO:381). The IgE CH2 CH3 CH4 region was inserted into thedigested pD18 vector above using methods described in Example 39. Theconstruct was designated G28-1 scFv VHL11S IgECH2 CH3 CH4 (SEQ IDNOs:379 and 383).

Example 36 Characterization of 2H7 scFv Ig Mutant Fusion Proteins

FIG. 54 illustrates the binding capacity of purified 2H7 scFv Igconstructs to CD20+CHO cells. The proteins were transfected into stableCHO cells according to methods described in Example 2. Binding wasdetermined using flow cytometry according to the methods described inExample 2. The graph in FIG. 54 illustrates that these proteins retainbinding function to CD20 with altered connecting regions. Comparativeresults were obtained in each of the 2H7 scFv VHL11S mutants with eachtype of altered connecting region (results omitted).

The ability of 2H7scFv-Ig constructs with mutated connecting regions tokill CD20 positive cells in the presence of peripheral blood mononuclearcells (PBMC) through ADCC was tested by measuring the release of ⁵¹Crfrom labeled BJAB cells in a 4 hr. assay using 100:1 ratio of PBMC toBJAB cells. The results shown in FIG. 55 indicate that 2H7scFv-Igmutants can mediate antibody dependent cellular cytotoxicity (ADCC),since the release of ⁵¹Cr was significantly higher in the presence ofboth PBMC and 2H7scFv-Ig than in the presence of either PBMC or2H7scFv-Ig alone. Comparative results were obtained in each of the 2H7scFv VHL11S mutants with each type of altered connecting region (resultsomitted).

The ability of 2H7scFv-Ig mutant fusion proteins to kill CD20 positivecells in the presence of complement was tested using B cell lines Ramostarget cells. Rabbit complement was purchased from Pel-Freez (Rogers,Ak.), and was used in the assay at a final concentration of 1/10.Purified 2H7scFv-Ig was incubated with B cells and complement for 45minutes at 37° C., followed by counting of live and dead cells by trypanblue exclusion. The results in FIG. 56 show that 2H7scFv-Ig mutants inthe presence of rabbit complement lysed B cells expressing CD20.

Example 37 Comparative Binding of IgA, IgG, and IgE 2H7 scFv Constructs

Binding capacity of Ig constructs IgA, IgG and IgE were measured usingflow cytrometry according to the methods described in Example 2, using acommercially available (Caltag) second step specific for each Ig tail.The results in FIG. 57 show that all the IgE constructs were able tobind CD20+CHO cells comparable to the binding abilities of IgG and IgA.These results also demonstrate that the IgE constructs were detectedwith the IgE second step, but not the IgA or IgG second step.

Example 38 Construction and Characterization of 2H7 VH L11S IgEConstructs

IgE tail RNA was isolated from SKO-007 cells(ATCC) using QIAGENQIAshredder homogenization and RNA minikits Random-primed cDNA wasgenerated according to the usual protocol, with 4 microliters RNA elutedfrom the QIAGEN columns. Human IgE from the beginning of CH1 through CH4(approximately 1.2 kb) was isolated by PCR amplification of 5microliters cDNA, with an amplification profile of 94 C, 60 sec; 72 C, 2minutes for 35 cycles, and the following primers:

5′ primer: (SEQ ID NO: 586) 5′-ggatccacccgctgctgcaaaaacattccctccaatgccacctccgtgac-3′ 3′ primer: (SEQ ID NO: 587)5′-tcatttaccgggatttacagacaccgctcgctggacg gtctgtgaggggctcgctgc-3′

PCR fragments were ligated into PCR 2.1-TOPO vector, and transformantsscreened for inserts of the correct size by digestion with EcoRIaccording to the methods described in Example 1. One of the clones withthe correct sequence from was used as template to amplify the CH2-CH4domains with appropriate restriction sites attached for subcloning assoluble or cell surface (ORF) forms. The following primers were usedwith an amplification profile of 94 C, 60 sec; 55 C, 60 sec; 72 C, 2min; for 35 cycles to amplify a fragment of approximately 950 bp:

5′ primer: (attaches BclI site to 5′ end of CH2 domain of IgE) (SEQ IDNO: 588) 5′-gttgttgatcacgtctgctccagggacttcacc-3′ 3′ primer: (attachesstop codon and XbaI site to 3′ end of CH4 of IgE) (SEQ ID NO: 589)5′-gttgtttctagattatcatttaccaggatttacagacaccgctcgctg-3′ 3′ primer:(attaches SfuI and BamHI to 3′ end of CH4 without a stop codon) (SEQ IDNO: 590) 5′-gttgttttcgaaggatccgctttaccagatttacagacaccgctcgctg-3′

The IgE CH2CH3CH4 tail with a stop codon was digested with BclI and XbaIand inserted into a pD18 vector that contains 2H7 VHL11S scFv. Thisconstruct was designated 2H7 IgECH2CH3CH4. The polynucleotide sequenceis provided in SEQ ID NO:128, and the encoded polypeptide sequence isprovided in SEQ ID NO:96. The IgE CH2CH3CH4 tail with no stop codon(ORF) was digested with BclI and SfuI and inserted in into a pD18 vectorthat contains 2H7 VHL11S scFv. This construct was designated 2H7IgECH2CH3CH4(ORF).

The human IgE was also amplified as a fragment missing both CH1 and CH2domains, with only the CH3 and CH4 domains attached to the human IgG1hinge. Sequential PCR reactions using overlapping 5′ oligonucleotideswere used to attach the IgG1 hinge to the CH3 domain of human IgE.Primers for the first step of the PCR reaction:

5′ Primer: (SEQ ID NO: 591)5′-actcacacatccccaccgtccccagcatccaacccgagaggggtga gc-3′

Primers for the second step of the PCR reaction:

5′ primer: (SEQ ID NO: 592)5′-tctgatcaggagcccaaatcttctgacaaaactcacacatccc caccg-3′ 3′ primer: (SEQID NO: 593) 5′-gttgtttctagattatcatttaccaggatttacagacaccgct cgctg-3′

The PCR product was digested with EcoRI and sequenced according to themethods described in Example 1. Positive clones were inserted into pD18plasmid containing 2H7 VHL11S scFv (SSS-S) H. The construct wasdesignated 2H7 VHL11S scFv (SSS-S) H IgE CH3CH4. The polynucleotidesequence is provided in SEQ ID NO:227, and the encoded polypeptidesequence is provided in SEQ ID NO:228.

Binding capacity of 2H7 scFv VH L 11S IgECH2 CH3 CH4, was measured usingflow cytometry, essentially according to Example 2. The protein waspurified using MEP HyperCel, (Cipergen, Catalog #12035-010,Lot#200920/0271) chromatography resin and Hydrophobic Charge InductionChromatography (HCIC). HCIC absorbent is a high capacity, highselectivity, absorbent designed for capture and purification ofmonoclonal and polyclonal antibodies from various sources including cellculture supernatants. Columns were packed with a 10 ml bead volume ofMEP Hypercel, and equilibrated with PBS, pH 7.4 containing 0.1% NaN3.Approximately 1 liter of 2H7 scFv VHL11S IgE CH2CH3CH4 CHO culturesupernatant was then run over the column. A series of citrate buffersranging from pH 3-6 were prepared for elution of the fusion protein. Thecolumn was washed in PBS. Protein was eluted in fifteen 1 ml fractionsat pH6, 5, and 4. A final 15 ml fraction was collected at pH 3.5.Aliquots from each fraction were analyzed for A280 and were alsosubjected to SDS-PAGE, loading roughly 10 micrograms/well based on theA280 reading. The results of these two analyses indicated that the bulkof the protein did not elute in citrate buffers at the higher pH, buteluted at pH4, and the post elution wash at pH3.5 also containedsignificant amounts of protein.

The ability of these 2H7 VH L11S IgE purified proteins to bind CD20+CHOcells was determined using flow cytometry according to the methodsdescribed in Example 2 using FITC-conjugated goat-anti-human IgE. FIG.58A illustrates that both purified proteins are able to bind CD20+CHOcells.

The ability of these 2H7 VH L11S IgE purified proteins to mediate ADCCagainst BJAB target cells with PBMC effectors was measured according tothe methods described in Example 2. FIG. 58B illustrates that bothproteins were able to mediate ADCC at similar levels.

Example 39 Construction and Binding Capacity of scFv VH L11S Mutantswith Mouse IgA and IgE Tail Regions

Murine IgA was cloned from murine spleen RNA using essentially the samemethods used to clone the human IgE tails in Example 38. The PCRreactions were performed with a 94 C 60 sec; 52 C 60 sec; 72 C 2 minamplification profile for 35 cycles. The PCR primers used to cloneCH1-CH4 regions were:

5′ primer: (SEQ ID NO: 594) 5′-atctgttctcctcctactactcctcctccacct-3′3′ primer: (SEQ ID NO: 595)5′-tcagtagcagatgccatctccctctgacatgatgacagacacgc t-3′PCR primers used to delete the CH1 region:

5′ primer: (SEQ ID NO: 596)5′-gttgttgatcacatctgttctcctcctactactcctcctccacct-3′ 3′ primer with astop codon, XbaI site at end of Ig tail, and the T4 mutation in CH3region: (SEQ ID NO: 597)5′-gttgtttctagattatcaatctccctctgacatgatgacagacac-3′ 3′ primer for theORF, a SfuI and BamHI sites, and T4 mutation in the CH3 region: (SEQ IDNO: 598) 5′-gttcttcgaaggatccgcatctccctctgacatgatgac-3′

The mouse IgACH2 T4-CH3 tail with a stop codon was digested with BclIand XbaI and inserted into a pD18 vector that contains 2H7 VHL11S scFvand the IgAH. This construct was designated 2H7 VHL11S scFv IgAH mIgACH2T4-CH3. The polynucleotide sequence is provided in SEQ ID NO:253, andthe encoded polypeptide sequence is provided in SEQ ID NO:25.

The mouse IgACH2 T4-CH3 tail with no stop codon (ORF) was digested withBclI and SfuI and inserted in into a pD18 vector that contains 2H7VHL11S scFv and IgAH. This construct was designated 2H7 VHL11S scFv IgAHmIgACH2 T4-CH3 (ORF). The polynucleotide sequence is provided in SEQ IDNO:255, and the encoded polypeptide sequence is provided in SEQ IDNO:256.

Murine IgE was cloned murine IgE La2 (ATCC) RNA essentially accordingthe methods described in Example 38. The PCR reactions were performedwith a 94 C 60 sec; 52 C 60 sec; 72 C 2 min amplification profile for 35cycles. Initial PCR primers used to clone the CH1-CH4:

5′ primer: (SEQ ID NO: 599) 5′-tctatcaggaaccctcagctctaccccttgaagccctg-3′3′ primer: (SEQ ID NO: 600)5′-gttgtttctagattatcaggatggacggagggaggtgttaccaa ggct-3′PCR primers to remove the CH1 region:

5′ primer: (SEQ ID NO: 601)5′-gttgttgatcacgttcgacctgtcaacatcactgagcccacc-3′ 3′ primer with stopcodon and XbaI site: (SEQ ID NO: 602)5′-gttgtttctagattatcaggatggacggagggaggtgttaccaaggct-3′ 3′ primer ORF,SfuI and Bam HI: (SEQ ID NO: 603)5′-gttgttttcgaaggatccgcggatggacggagggaggtgtta-3′

The mouse IgE CH2CH3CH4 tail with a stop codon was digested with BclIand XbaI and inserted into a pD18 vector that contains 2H7 VHL11S scFv.This construct was designated 2H7 VHL11S mIgECH2CH3CH4. Thepolynucleotide sequence is provided in SEQ ID NO:249, and the encodedpolypeptide sequence is provided in SEQ ID NO:250.

The mouse IgE CH2CH3CH4 tail with no stop codon (ORF) was digested withBclI and SfuI and inserted in into a pD18 vector that contains 2H7VHL11S scFv. This construct was designated 2H7 VHL11S scFvmIgECH2CH3CH4(ORF). The polynucleotide sequence is provided in SEQ IDNO:249, and the encoded polypeptide sequence is provided in SEQ IDNO:250.

Binding capacity of 2H7 VH L11S mIgE and mIgA (with mouse tail regions)to CD20+CHO cells were also measured by flow cytometry according to themethods described in Example 2 using commercially available IgE or IgAsecond step reagents (Caltag). Each point in FIG. 59 represents the meanof a population with brightness corrected by subtracting the binding ofthe second step alone. This figure illustrates that these constructshave the ability to bind CD20+CHO cells.

Example 40 HPLC Profiles of 2H7 scFv Ig Mutant Fusion Proteins

HPLC analysis of purified 2H7 scFv-Ig mutant fusion proteins withaltered connecting and CH3 regions. Each protein was purified by ProteinA affinity chromatography from supernatants of transfected COS or CHOcells. Twenty-five to fifty micrograms of each sample was run at 1ml/min in PBS on a TSK-GEL G3000SW_(XL) 30 cm column (Tosoh Biosep,Stuttgart, Germany). The arrow near the beginning of each profilerepresents the sample injection point. Gel filtration standards(Bio-Rad) included thryoglobulin (670 kDa), gamma globulin (158 kDa),ovalbumin (44 kDa), myoglobin (12.5 kDa), and vitamin B-12 (1.35 kDa).Standards were run at the beginning and end of each experiment.Migration positions of standards are shown and did not vary betweenexperiments. (FIGS. 60-62)

Example 41 Binding Capacity of 2H7 VH L11S Mutant Fusion Proteins

Binding effects of the CH3 mutant were compared to the non-mutated CH3fusion protein. The constructs were transfected into COS cells andpurified from the supernatant using protein A column purificationtechniques described in Example 2. FIG. 63 illustrates the differentialeffects of CH3 mutations on binding using flow cytometry according tothe methods described in Example 2. This figure illustrates that somebinding ability is lost when the double point mutation is introduced inthe CH3 region.

Binding of fluorescein isothiocyanate (FITC) conjugated 2H7 VH L11Smutant fusion proteins was determined using flow cytometry. A 1 mg/mlsolution of FITC was prepared in DMSO. Fusion proteins were dialyzed inpH 9.3 bicarbonate buffer overnight at 4 C in a volume of 2 liters.Concentration of the protein was adjusted to 1-5 mg/ml prior toconjugation. A series of Falcon 5 ml tubes was set up with varying FITCto protein ratios, ranging from 15-60, but minimally ratios of 20 and40. Conjugation reactions were incubated at 37 C for 30 minutesprotected from light. Fluoresceing labeled protein was separated fromfree fluorescein on a 2 ml Sephadex G-25 column equilibrated with PBS,0.5 M NaCl, and 1% NaN3. The fluorescein labeled protein eluted from thecolumn first and was collected in a 5 ml tube. The degree of labelingwas determined by measuring the absorbance of the diluted conjugate at280 and 494 nm, and utilizing the formulas provided by technicalservices at Molecular Probes (Eugene, Oreg.). Data has been correctedfor FITC: protein ratio. FIG. 64 illustrates that these constructs donot lose binding capacity when conjugated to a fluorescent marker.

Purified 2H7 VH L11S constructs were run on a non-reducing SDS gelaccording to the methods described in Example 2. The migration patternsare presented in FIG. 65.

Example 42 Characterization of 2H7 scFv VH L11S (CSC-S) H WCH2 WCH3 inLec13 CHO Cells

2H7 scFv VH L11S (CSC-S) H WCH2 WCH3 were transiently transfected andexpressed in Lec13 CHO cells. Lec13CHO cells were used as the mammaliancell hosts for either the 2H7 scFv VHS11 hIgG1 (CSS-S) H WCH2 WCH3 and(CSC-S) H WCH2 WCH3 expressing plasmids in side-by-side transfections.All transfections were performed in 100 mm tissue culture dishes. Cellswere transfected when approximately 90% confluent using lipofectamine2000 (Invitrogen, Catalog #: 11668-027, 0.75 ml), followingmanufacturer's instructions. Both cell lines were grown in the presenceof serum to promote and maintain adherence to the cell culture dishes,simplifying transfection manipulations, washes, and supernatantharvests. DNA: lipofectamine complexes were allowed to form in theabsence of serum and antibiotics, following the suggestedprotocol/conditions recorded in the product insert. Culture supernatantswere harvested 72 hours after transfection, and then again 72 hoursafter the first harvest. Fusion protein from the two CHO sources wasisolated by protein A purification as previously described and used inCD20 binding and ADCC assays.

The ability of mutated fusion protein to mediate ADCC in CD20 positivecells was determined using the methods described in Example 2.Constructs expressed in Lec13 CHO cells exhibited better binding to CD20CHO target cells and also showed significantly improved activity in ADCCassays relative to the CHO DG44 derived proteins at equivalentconcentrations as illustrated in FIG. 67.

Example 43 Construction of High and Low Affinity CD16 Alleles

The low(V) and high(F) affinity alleles at position 158 of the humanCD16 extracellular domain were cloned from cDNA derived from human PBMCusing PCR assay. PCR reactions used random primed cDNA made from PBMCstimulated for 3 days with immobilized anti-CD3 antibody (64.1) prior toharvest. PCR reactions included 2, 4, 6 or 8 microliters of cDNA, eachprimer at 25 pmol, and an amplification profile of 94 C 60 sec; 55 C 60sec; 72 C 2 min, for 35 cycles. The PCR primers are listed below:

5′ primer-no leader peptide: (SEQ ID NO: 604)5′-GTTGTTACCGGTGCAATGCGGACTGAAGATCTCCC AAAGGCTGTG-3′ 3′ antisenseprimer: (SEQ ID NO: 605)5′-GTTGTTTGATCAGCCAAACCTTGAGTGATGGTGATGTTCACA-3′

Positive clones were sequenced, and inserted into a vector containing anefficient leader peptide and the (SSS-S)H P238SCH2 WCH3 human IgG tail.Two different versions of the CD16 ED fusion proteins were expressed.The first contained the F158 (high affinity) and the second containedthe V158 (low affinity) allele. Constructs were cloned into a (SSS-S)HP238S CH2 WCH3 pD18 plasmid and expressed in COS and CHO cells aspreviously described in examples 1 and 10. CHO clones were screened forexpression using an IgG sandwich ELISA to determine relative expressionlevels of the fusion proteins in the culture supernatant using thefollowing protocol: Immulon 1V plates were coated at 4 C with 0.4microgram/ml goat anti-human IgG (mouse Adsorbed), (CalTag, Catalog#H10500) in PBS buffer. Plates were then blocked in PBS/1.5% nonfat milkat 4 C overnight. Plates were washed three times in PBS/0.1% Tween 20,then incubated with 100 microliters dilution series from CHO cloneculture supernatants at room temperature for 3 hours. Four dilutions perclone were added to successive wells, diluting in 5 fold increments from1:5 to 1:375. In addition a standard curve was derived using CTLA4 hIgG1(SSS-S)H P238SCH2 WCH3 as a concentration standard. The dilution seriesutilized 5 fold dilutions starting at 0.5 micrograms/ml; a second set of8 wells was used to make a 2-fold dilution series starting at 0.34micrograms/ml. Plates were washed 3 times in PBS/0.1% Tween 20, andincubated with goat anti-human IgG, conjugated to horseradish peroxidase(GAH IgG-HRP) at 1:5000, in PBS/0.5% BSA for 1 hour. Plates were washedfour times with PBS/0.1% Tween 20, then TMB chromagen substrate(BD-Pharmingen) was added for 10 minutes, and reactions stopped byaddition of 100 microlites 1N sulfuric acid. Plates were then read at415 nm on a SpectraCount plate reader. Concentrations of fusion proteinwere estimated by comparison of the ODs in the linear range to theCTLA4Ig standard curve run on each plate.

Proteins were purified using Protein A purification and were directlyconjugated to fluorescein isothiocyanate (FITC) as described in Example42. These proteins were run out on SDS gels under reduced and nonreducedconditions according to the methods described in Example 2. Themigration of these proteins is presented in FIG. 68.

The ability of the high and low CD16 alleles to bind 2H7 VH111S(CSC-S) HWCH2 WCH3 or bind 2H7 VH111S (SSS-S) H (P238S)CH2 WCH3 expressed on thecell surface of CD20+CHO target cell is determined using flow cytometryaccording to the methods described in Example 2. The results in FIG. 69demonstrate both the high and low affinity alleles were able to bind 2H7VHL11S (CSC-S)H WCH2 WCH3 (SEQ ID NO:246) and lost some bindingcapabilities when the P238S mutation was introduced into the CH2 regionof the construct (SEQ ID NOs:417 and 418).

Example 44 Mammalian Display System

FIG. 70A diagrams how FITC conjugates of FcRIII (CD16) Soluble Fusionproteins bind to 2H7 scFv-Ig constructs that are attached to CD20expressed by CHO cells. The CD16 binding to a scFv-Ig provides ascreening tool for detecting changes in CD16 binding to an alteredscFv-Ig constructs containing targeted or site-specific mutations.Changes in CD16 binding properties may be changes in binding of eitherCD16 high affinity protein (158 F) or CD16 low affinity protein (158 V)or both.

A schematic representation of such a screening process is diagrammed inFIG. 70B, where scFv-Ig constructs are displayed on the cell surface ofmammalian cells. The scFv-Ig molecules in this Example are displayed onthe cell surface because they contain a transmembrane domain anchor.These molecules may represent a single scFv-Ig construct or may beintroduced into a population of mammalian cells as a library of suchmolecules. Transfected cells with altered binding properties can then bepanned, sorted, or otherwise isolated from other cells by altering thestringency of the selection conditions and using CD16 fusion proteins asthe binding probe. Cells that express scFv-Ig molecules with alteredbinding to either CD16 high affinity allele (158 F) or CD16 low affinityallele (158V) or both can be isolated. For example, this display systemcan be used to create a library of mutated Ig tails with short stretchesof CH2 sequence replaced with randomized oligonucleotides or possiblyrandomization of a single residue with all possible amino acidsubstitutions, including synthetic amino acids. Once such a library isconstructed, it can be transfected into COS cells by methods well knownin the art. Transfectants can then be bound to the labeled CD16constructs, and panned or sorted based on their relative bindingproperties to multiple allelotypes/isoforms. Panned cells are harvested,and the plasmid DNA is isolated and then transformed into bacteria. Thisprocess may be repeated iteratively multiple times until single clonesare isolated from the mammalian host cells (see Seed B and Aruffo A,PNAS 84:3365-3369 (1987) and Aruffo A and Seed B, PNAS 84: 8573-8577(1987)). One such use of this type of screening system would be toisolate Ig tails which bind equally well to both the high and lowaffinity alleles of CD16 with the goal of improving effector functionsmediated by scFv-Ig constructs in multiple subpopulations of patients.Ig tails with altered binding properties to other Fc receptors can alsobe selected using the display system described. Other display systemsfor example those that use bacteriophage or yeast are not suitable forselection of Ig tails with altered FcR binding properties because of therequirement for glycosylation in the Ig CH2 domain that would not occurin non-mammalian systems.

This system is also useful for selection of altered scFv-Ig moleculesthat will be produced at higher levels. In this Example, mammalian cellssuch as COS cells can be transfected with a library of scFv-Igconstructs in a plasmid that directs their expression to the cellsurface. COS cells that express the highest levels of the scFv-Igmolecules can be selected by techniques well known in the art (forexample panning, sterile cell sorting, magnetic bead separation, etc),and plasmid DNA is isolated for transformation into bacteria. Afterseveral rounds of selection single clones are isolated that encodescFv-Ig molecules capable of high level expression. When the isolatedclones are altered to remove the membrane anchor and then expressed inmammalian cells, the scFv-Ig constructs will be secreted into theculture fluid in high levels. This reflects the common requirement ofsecreted glycoproteins and cell surface glycoproteins for a signalpeptide and processing through the golgi for expression, so thatselection for a molecule that illustrates an improvement in expressionlevels on the cell surface will also select for a molecule thatillustrates an improvement in levels of secreted protein.

Example 45 Characterization of G28-1 mAbs and scFvs

Ability of G28-1 mAbs and scFvs to induce apoptosis was measured bybinding Annexin V, using the methods described in Example 3. The resultsin FIG. 71 demonstrate that the Annexin V binding of G28-1 antibodiesand scFv is increased when treated together with 2H7 antibodies and scFvconstructs.

Example 46 Construction of FC2-2 scFv Constructs

Contrstruction of the FC2-2 (anti-CD16) scFv was performed using totalRNA isolated from the FC2-2 hybridoma and cloned using methods describedin Example 35. The polynucleotide sequence is provided in SEQ ID NO:337,and the encoded polypeptide sequence is provided in SEQ ID NO:338. Thespecific primers for the secondary PCR reaction are listed bellow. Thefollowing are primers for the light chain variable region:

5′ primer with HindIII site with no leader: (SEQ ID NO: 606)5′-GTTGTTAAGCTTGCCGCCATGGATTCAC AGGCCCAGGTTCTT-3′ 5′ primer with SalIsite and leader: (SEQ ID NO: 607) 5′-GTTGTTGTCGACATTGTGATGTCACAGTCTCCATCCTCCCTA-3′ 3′ primer: (SEQ ID NO: 608)5′-TCAGTGCTGATCATGAGGAGACGGTGACTGAGGTTCCT-3′

The following are primers for the heavy chain variable region:

5′ primer: (SEQ ID NO: 609)5′-TCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCGTCACAGGTGCAGTT GAAGGAGTCAGGA-3′3′ primer: (SEQ ID NO: 610)5′-ACCCGACCCACCACCGCCCGAGCCACCGCCACCTTTTATTTCCAGCT TGGTGCCACCTCCGAA-3′

A change from leucine to serine at position 11 in the heavy chainvariable region (Kabat numbering) was introduced in into the FC2-2 scFvby site-directed mutagenesis according to the methods described inExample 33. The scFv was attached to the (SSS-S) H WCH2 WCH3 IgG tailaccording to methods described in Example 33. The mutant is designatedFC2-2 scFv VH L11S (SSS-S) H WCH2 WCH3. The polynucleotide sequence isprovided in SEQ ID NO:345, and the encoded polypeptide sequence isprovided in SEQ ID NO:346.

Example 47 Construction of 5B9 scFv Constructs

Contrstruction of the 5B9 (anti-CD137) scFv was performed using totalRNA isolated from the 5B9 hybridoma and cloned using methods describedin Example 35. The polynucleotide sequence is provided in SEQ ID NO:361,and the encoded polypeptide sequence is provided in SEQ ID NO:362. Thespecific primers for the secondary PCR reaction are listed bellow. Thefollowing are primers for the light chain variable region:

5′ primer with HindIII site with no leader: (SEQ ID NO: 611)5′-GTTGTTAAGCTTGCCGCCATGAGGTTCT CTGCTCAGCTTCTG-3′ 5′ primer with SalIsite and leader: (SEQ ID NO: 612) 5′-GTTGTTGTCGACATTTGTGATGACGCAGGCTGCATTCTCCAATT-3′ 3′ primer: (SEQ ID NO: 613)5′-TCAGTGCTGATCAGAGGAGGACGGTGACTGAGGTTCCTTG-3′

The following are primers for the heavy chain variable region:

5′ primer: (SEQ ID NO: 614)5′-CGGGCGGTGGTGGGTCGGGTGGCGGCGGATCGTCACAGGTGCAGCTG AAGCAGTCAGGA-3′3′ primer: (SEQ ID NO: 615)5′-CCCGACCCACCACCGCCCGAGCCACCGCCACCCTTCAGCTCCAGCTT GGTGCCAGCACC-3′

A change from leucine to serine at position 11 in the heavy chainvariable region (Kabat numbering) was introduced in into the 5B9 scFv bysite-directed mutagenesis and attached to (SSS-S) H WCH2 WCH3 accordingto the methods described in Example 33. This construct was designated5B9 scFv VHL11S (SSS-S) H WCH2 WCH3. The polynucleotide sequence isprovided in SEQ ID NO:365, and the encoded polypeptide sequence isprovided in SEQ ID NO:366.

Example 48 Construction of UCHL1 scFv Constructs

Contrstruction of the UCHL1 (anti-CD45RO) scFv was performed using totalRNA isolated from the UCHL1 hybridoma and cloned using methods describedin Example 35 The polynucleotide sequence is provided in SEQ ID NO:351,and the encoded polypeptide sequence is provided in SEQ ID NO:352. Thefollowing are primers for the light chain variable region:

5′ primer with HindIII site: (SEQ ID NO: 616)5′-GTTGTTAAGCTTGCCGCCATGAAGTTGCCTGTTAGGCTG TTGGTGCTG-3′ 3′ primer withSac site: (SEQ ID NO: 617) 5′-AGAGCTCCCACCTCCTCCAGATCCACCACCGCCCGAGCCACCGCCATCTTTGATTTCCAGCTTGGT-3′

The following are primers for the heavy chain variable region:

5′ primer: (SEQ ID NO: 618)5′-TTTCAGAGTAATCTGAGAGCTCCCACCTCCTCCAGATCCACCACCGC CCGA-3′ 3′ primer:(SEQ ID NO: 619) 5′-TCAGTGCTGATCATGCAGAGAGACAGTGACCAGAGTCCC-3′

A change from leucine to serine at position 11 in the heavy chainvariable region (Kabat numbering) was introduced in into the 5B9 scFv bysite-directed mutagenesis and connected to a (SSS-S) HWCH2 WCH3according to the methods described in Example 33. The mutant isdesignated UCHL1 scFv VH L11S (SSS-S) H WCH2 WCH3. The polynucleotidesequence is provided in SEQ ID NO:359, and the encoded polypeptidesequence is provided in SEQ ID NO:360.

Example 49 L6 VHL11S scFv (SSS-S) H WCH2 WCH3

A change from leucine to serine at position 11 in the heavy chainvariable region (Kabat numbering) was introduced in into the L6 scFv(SSS-S) H WCH2 WCH3 (constructed according to methods described inExample 106) by site-directed mutagenesis according to the methodsdescribed in Example 33. The L6scFvIg (SSS-S) H WCH2 WCH3 pD18 plasmidwas used as template. Positive clones were inserted into the pD18plasmid containing (SSS-S) H WCH2 WCH3 according to methods described inExample 33. The mutant is designated L6 scFv VH L11S (SSS-S) H WCH2WCH3. The polynucleotide sequence is provided in SEQ ID NO:415, and theencoded polypeptide sequence is provided in SEQ ID NO:416. PCR primersare listed bellow:

5′ Primer with PstI restriction site: (SEQ ID NO: 620)5′-ggcggatctctgcagatccagttggtgcagtctggacctgagtcga agaagcctggagag-3′3′ Primer: (SEQ ID NO: 621) 5′-ggacagtgggagtggcacc-3′

Example 50 Construction of HD37 scFv VHL11S Construct

A change from leucine to serine at position 11 in the heavy chainvariable region (Kabat numbering) was introduced in into the HD37 scFvby site-directed mutagenesis according to the methods described inExample 33. The HD37 scFv (SSS-S)H WCH2 WCH3 pD18 plasmid was used as atemplate. Positive clones were inserted into the pD18 plasmid containing(SSS-S) H WCH2 WCH3 according to methods described in Example 33. Themutant is designated HD37 scFv VH L11S (SSS-S) H WCH2 WCH3. Thepolynucleotide sequence is provided in SEQ ID NO:401, and the encodedpolypeptide sequence is provided in SEQ ID NO:402. PCR primers arelisted bellow:

5′ primer: (SEQ ID NO: 622)5′-caggttcagctgcagcagtctggggctgagtcggtgaggcctgg-3′ 3′ primer: (SEQ IDNO: 623) 5′-ggaggattcgtctgcagtcagagtggc-3′

Example 51 2H7 scFv VHL11S Constructs

Additional 2H7 VHL11S constructs were made with different connectingregions. The pD18 2H7 scFv VHL11S (SSS-S) H WCH2 WCH3 vector wasdigested with BclI and XbaI to remove the connecting region, CH2 andCH3. Theses were replaced with each different connecting region, CH2 andCH3 according to the methods described in Example 13. The new constructswere designated: 2H7scFv VHL11S (CSS-S) H WH2 WH3 (SEQ ID NO:371),2H7scFv VHL11S (CSC-S) H WH2 WH3 (SEQ ID NO:245). 2H7 scFv VHL11S wasalso attached to an IgA connecting region, CH2, CH3 and an IgE CH2, CH3,CH4. The pD18 2H7 scFv VHL11S (SSS-S) H WCH2 WCH3 plasmid was digestedusing methods above to remove the connecting region CH2, and CH3. TheIgA regions were inserted using methods described in Example 13. Theconstruct was designated 2H7 scFv VHL11S IgAH IgACH2 T4CH3 (SEQ IDNOs:251 and 253). The IgE CH2 CH3 CH4 region was inserted into thedigested pD18 vector above using methods described in Example 39. Theconstruct was designated 2H7 scFv VHL11S IgECH2 CH3 CH4 (SEQ ID NO:247).

Example 52 2H7 scFv VH L11S (CSC-S) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is connected to a human IgG1 connectingregion, CH2 and CH3 region, where in the second cysteine and the prolinein the connecting region have been changed to serines (SSS-S) asdescribed in Example 32. The polynucleotide sequence is provided in SEQID NO:245, and the encoded polypeptide sequence is provided in SEQ IDNO:246.

Example 53 2H7 scFv VH L11S IgE CH2 CH3 CH4

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is attached to a human IgE constantregion containing CH2, CH3 and CH4 as described in Example 38. Thepolynucleotide sequence is provided in SEQ ID NO:247, and the encodedpolypeptide sequence is provided in SEQ ID NO:248.

Example 54 2H7 scFv VH L11S mIgE CH2 CH3 CH4

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is attached to a mouse IgE constantregion containing CH2, CH3 and CH4 as described in Example 39. Thepolynucleotide sequence is provided in SEQ ID NO:249, and the encodedpolypeptide sequence is provided in SEQ ID NO:250.

Example 55 2H7 scFv VH L11S mIgAH WIgACH2 T4CH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is attached to a connecting region fromhuman IgA as described in Example 5. This connecting region is attachedto a mouse IgA constant region consisting of a wild type CH2 region anda mutated CH3 region where there is a truncation of 4 amino acidresidues prior to the 3′ stop codon as described in Example 39. Thepolynucleotide sequence is provided in SEQ ID NO:253, and the encodedpolypeptide sequence is provided in SEQ ID NO:254.

Example 56 2H7 scFv VH L11S (SSS-S) H K322S CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is connected to a mutated human IgGconnecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.The connecting region is attached to a mutated IgG CH2 region and a wildtype IgG CH3 region. The K322S mutation in the CH2 region is at aresidue 322, where a Lysine has been changed to a serine usingoverlapping PCR assay. An (SSS-S) H WCH2 WCH3 IgG1 template in the pD18vector was used for PCR amplification, to create (SSS-S) H derivativescontaining these CH2 mutations. PCR reactions used a cycling profile of94 C, 30 sec; 55 C, 30 sec; 72 C, 30 sec, for 37 cycles to complete thereactions. This IgG1 derivative was constructed by using sequential PCRreactions with overlapping oligonucleotides in the primary and secondaryreactions. The primary amplification primers introduced the mutation(s),but deleted one end of the Fc domain. Secondary reaction primersreattached these ends using overlapping primers. The first overlappingprimer was added at the beginning of the PCR, the reactions allowed toproceed for 12 cycles, paused and then the second overlapping primeradded to the reactions followed by 25 more cycles to complete theoverlap extension PCR reactions.

Primers for the first PCR reaction:

5′ Primer: (SEQ ID NO: 624) 5′-ggagatggttttctcgatgggggctgggagggctttgttggagaccgagcacttgtactcc-3′ 3′ primer: (SEQ ID NO: 625)5′-ggacagtgggagtggcacc-3′

PCR products were cloned into TOPO vector and sequenced forverification. Positive vectors were used as templates for the secondoverlapping PCR reaction.

5′ primer: (SEQ ID NO: 626) 5′-ccgtctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagc-3′ 5′ primer overlapping primer: (SEQ ID NO:627) 5′-tccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacc-3′ 3′ primer: (SEQ ID NO: 628)5′-caggaaacagctatgac-3′

PCR product was cloned into TOPO vector and sequenced. Thepolynucleotide sequence is provided in SEQ ID NO:263, and the encodedpolypeptide sequence is provided in SEQ ID NO:264.

Example 57 2H7 scFv V11 L11S (CSS-S) H K322S CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is attached to a mutant IgG connectingregion, where the second and third cysteines have been changed toserines and the proline has been changed to serine (CSS-S), according tomethods described in Example 13. The connecting region is attached to amutated IgG CH2 region and a wild type IgG CH3 region. The K322Smutation in the CH2 region is at a residue 322, where a Lysine has beenchanged to a serine using overlapping PCR assay. region is attached to amutated IgG CH2 region and a wild type IgG CH3 region. The mutation inthe CH2 region was added by overlapping PCR reaction essentiallyaccording to Example 57, with (CSS-S) H WCH2 WHC3 IgG1 pD18 vector as atemplate in the first PCR reaction and different primers in the secondPCR reaction, which are listed below.

5′primer: 5′-ccgtctctgatcaggaccccaaatcttgtgacaa (SEQ ID N0: 629)aactcacacatccccaccgtccccagc-3′ 5′overlapping primer:5′-tccccaccgtccccagcacctgaactcctggggg atcgtcagtcttcctcttccccccaaaacc-3′(SEQ ID NO: 630) 3′primer: 5′-caggaaacagctatgac-3′ (SEQ ID NO: 628)

PCR products were cloned into the TOPO vector and sequenced. Thepolynucleotide sequence is provided in SEQ ID NO:267, and the encodedpolypeptide sequence is provided in SEQ ID NO:268.

Example 58 2H7 scFv VH L11S (SSS-S) H P331S CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is connected to a mutated human IgG1connecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.The connecting region is attached to a mutated IgG1 CH2 region and awild type IgG1 CH3 region. The mutation P331S mutation in the CH2region, where the proline at residue 331 has been changed to a serine,was incorporated using a single PCR reaction, using a (SSS-S) H WCH2WCH3 pD18 template and a cycling profile of 94 C, 30 sec; 55 C, 30 sec;72 C, 30 sec, for 37 cycles. The specific primers for reaction arelisted below.

5′ primer: 5′ccgtctctgatcaggagcccaaatcttctgacaaaac (SEQ ID NO: 631)tcacacatccccaccgtccccagc-3′ 3′ Primers:5′-gcagggtgtacacctgtggttctcggggctgccctt (SEQ ID NO: 632)tggctttggagatggttttctcgatggaggctggg agg-3′

The polynucleotide sequence is provided in SEQ ID NO:273, and theencoded polypeptide sequence is provided in SEQ ID NO:274.

Example 59 2H7 scFv V11 L11S (CSS-S) H P331S CH2 WCH3

This binding region is attached to a mutant IgG connecting region, wherethe second and third cysteines have been changed to serines and theproline has been changed to serine (CSS-S), according to methodsdescribed in Example 13. The connecting region is attached to a mutatedIgG CH2 region and a wild type IgG CH3 region. The mutation P331Smutation in the CH2 region, where the proline at residue 331 has beenchanged to a serine, was incorporated using a single PCR reaction, usinga (CSS-S) H WCH2 WCH3 pD18 template and a cycling profile of 94 C, 30sec; 55 C, 30 sec; 72 C, 30 sec, for 37 cycles. The specific primers forreaction are listed below.

5′primer: 5′-ccgtctctgatcaggaccccaaatcttgtgacaaaa (SEQ ID NO: 633)ctcacacatccccaccgtccccagc-3′ 3′Primer:5′-gcagggtgtacacctgtggttctcggggctgccctt (SEQ ID NO: 634)tggctttggagatggttttctcgatggaggctggg agg-3′

The polynucleotide sequence is provided in SEQ ID NO:275, and theencoded polypeptide sequence is provided in SEQ ID NO:276.

Example 60 2H7 scFv VH L11S (SSS-S) H T256N CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is connected to a mutated human IgGconnecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.The connecting region is attached to a mutated IgG CH2 region and a wildtype IgG CH3 region. The T256N mutation in the CH2 region, the threonineat residue 256 has been changed to an asparagine, using the overlappingPCR methods described in Example 56. The specific primers are listedbelow.

Primers for the first PCR reaction:

5′ primer: 5′ttcctcttccccccaaaacccaaggacaccctcatga (SEQ ID NO: 635)tctcccggaaccctgaggtcac-3′ 3′ primer: 5′-ggacagtgggagtggcacc-3′ (SEQ IDNO: 636)

PCR product cloned into TOPO vector and sequenced. This product was usedas the template in the second PCR reaction. Primers for the second PCRreaction:

5′primer: 5′ccgtctctgatcaggagcccaaatcttctgacaaaac (SEQ ID NO: 637)tcacacatccccaccgtccccagc-3′ 5′overlapping primer:5′-tccccaccgtccccagcacctgaactcctgggggga (SEQ ID NO: 638)tcgtcagtcttcctcttccccccaaaacc-3′ 3′primer: 5′-caggaaacagctatgac-3′ (SEQID NO: 628)

The polynucleotide sequence is provided in SEQ ID NO:281, and theencoded polypeptide sequence is provided in SEQ ID NO:282.

Example 61 2H7 scFv VH L11S (SSS-S) H RTPE/QNAK (255-258) CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is connected to a mutated human IgGconnecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.The connecting region is attached to a mutated IgG CH2 region and a wildtype IgG CH3 region. The RTPE/QNAK mutation in the CH2 region, whereresidues 255-258 have been mutated from arginine, threonine, proline,glutamic acid to glutamine, asparagines, alanine and lysine,respectively, using the overlapping PCR reactions described in Example56. The specific primers are listed below.

PCR primers for the first PCR reaction:

5′ primer: 5′-ttcctcttccccccaaaacccaaggacaccctca (SEQ ID NO: 641)tgatctcccagaacgctaaggtcacatgc-3′ 3′ primer: 5′-ggacagtgggagtggcacc-3′(SEQ ID NO: 636)

The PCR product was cloned into TOPO vector, sequenced and used as atemplate for the second PCR reaction. The primers for the second PCRreaction:

5 primer: 5′ccgtctctgatcaggagcccaaatcttctgacaaaac (SEQ ID NO: 637)tcacacatccccaccgtccccagc-3′ 5′overlapping primer:5′-tccccaccgtccccagcacctgaactcctgggggga (SEQ ID NO: 638)tcgtcagtcttcctcttccccccaaaacc-3′ 3′primer: 5′-caggaaacagctatgac-3′ (SEQID NO: 628)

The polynucleotide sequence is provided in SEQ ID NO:289, and theencoded polypeptide sequence is provided in SEQ ID NO:290.

Example 62 2H7 scFv VH L11S (SSS-S) H K290Q CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is connected to a mutated human IgG1connecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.The connecting region is attached to a mutated human IgG1 CH2 region anda wild type human IgG1 CH3 region. The K290Q mutation in the CH2 region,where the Lysine at residue 290 has been changed to a Glutamine, using asingle PCR reaction according to the methods described in Example 58.The specific primers used in this reaction are listed below.

5′ primer: 5′ccgtctctgatcaggagcccaaatcttctgacaaaac (SEQ ID NO: 642)tcacacatccccaccgtccccagc-3′ 3′ primer: 5′-gctcccgcggctgtgtcttggc-3′ (SEQID NO: 645)

PCR products were cloned into TOPO and sequenced. The polynucleotidesequence is provided in SEQ ID NO:297, and the encoded polypeptidesequence is provided in SEQ ID NO:298.

Example 63 2H7 scFv VH L11S (SSS-S) H A339P CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is connected to a mutated human IgG1connecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.The connecting region is attached to a mutated human IgG1 CH2 region anda wild type human IgG1 CH3 region. The A339P mutation in the CH2 region,where the alanine at residue 339 has been changed to a proline, using asingle PCR reaction according to the methods described in Example 58.The specific primers used in this reaction are listed below.

5′ primer: 5′ccgtctctgatcaggagcccaaatcttctgacaaaac (SEQ ID NO: 644)tcacacatccccaccgtccccagc-3′ 3′ Primer:5′-ggaggtgggcagggtgtacacctgtggttctcgggg (SEQ ID NO: 647)ctgccctttgggtttggagatgg-3′

PCR products were cloned into TOPO and sequenced. The polynucleotidesequence is provided in SEQ ID NO:305, and the encoded polypeptidesequence is provided in SEQ ID NO:306.

Example 64 G28-1 scFv (SSS-S) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionmade according to methods described in Example 35. This binding regionis connected to a mutated human IgG1 connecting region where all of thecysteines and one proline have been changed to serines (SSS-S) accordingto methods described in Example 5. This connecting region is connectedto a wild type human IgG1 CH2 and CH3 region as described in Example 1.This construct has previously been referred to as G28-1-MHWTG1C andG28-1 scFv Ig, both have the same sequence as the abouve construct. Thepolynucleotide sequence is provided in SEQ ID NO:319, and the encodedpolypeptide sequence is provided in SEQ ID NO:320.

Example 65 G28-1 scFv IgAH WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionmade according to methods described in Example 35. This binding regionis connected to a human IgA connecting region and wild type human IgGCH2 and CH3 constant regions as described in Example 5. This constructhas previously been referred to as: G28-1-IgAHWTG1C. The polynucleotidesequence is provided in SEQ ID NO:321, and the encoded polypeptidesequence is provided in SEQ ID NO:322.

Example 66 G28-1 scFv VH L11S (SSS-S) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 region as described in Example 1. The polynucleotidesequence is provided in SEQ ID NO:323, and the encoded polypeptidesequence is provided in SEQ ID NO:324.

Example 67 G28-1 scFv VH L11S(CSS-S) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a mutanthuman IgG1 connecting region, where the second and third cysteines havebeen changed to serines and the proline has been changed to serine(CSS-S), according to methods described in Example 13. This connectingregion is attached to wild type human IgG1 CH2 and CH3 region asdescribed in Example 1. The polynucleotide sequence is provided in SEQID NO:325, and the encoded polypeptide sequence is provided in SEQ IDNO:326.

Example 68 G28-1 scFv VH L11S (CSC-S) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a mutanthuman IgG1 connecting region, where the second cysteine and the prolinehas been changed to serine (CSC-S), according to methods described inExample 32. This connecting region is attached to wild type human IgG1CH2 and CH3 region as described in Example 1. The polynucleotidesequence is provided in SEQ ID NO:327, and the encoded polypeptidesequence is provided in SEQ ID NO:328.

Example 69 G28-1 scFv VH L11S (SSC-P) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a mutanthuman IgG1 connecting region, where the first and second cysteines havebeen changed to serines (SSC-P), according to methods described inExample 13. This connecting region is connected to a wild type humanIgG1 CH2 and CH3 region as described in Example 1. The polynucleotidesequence is provided in SEQ ID NO:329, and the encoded polypeptidesequence is provided in SEQ ID NO:330.

Example 70 CTLA4 (SSS-S) H P238SCH2 WCH3

This construct has the extra cellular CTLA-4 binding region as describedin Example 14. This binding region is connected to a mutated human IgG1connecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.This hinge region is attached to a mutated human IgG1 CH2 region and awild type human IgG1 CH3 region. The P238S mutation, where a proline atresidue 238 was changed to a serine, was introduced using a PCR assay.PCR reactions were performed using random primed cDNA prepared fromhuman tonsil B cell RNA. PCR amplifications used an amplificationprofile of 94 C 4 min; [94 C 1 min; 55 C 1 min; 72 C 1 min; for 30cycles followed by a final extension step for 6 minutes at 72 C. PCRfragments were TOPO cloned and clones with EcoRI inserts ofapproximately 800 bp were sequenced as described in Example 1. Theprimers used for the PCR are listed below:

5′ primer: 5′-gttgttgatcaggagcccaaatcttctgacaaaact (SEQ ID NO: 648)cacacatctccaccgtccccagcacctgaactcctgggt ggaccgtcagtcttcc-3′ 3′ primer:5′gttgtttctagattatcatttacccggagacag-3′ (SEQ ID NO: 649)

This construct has previously been referred to as CTLA-4 IgG MTH (SSS)MTCH2WTCH3, which has the same sequence as the above construct. Thepolynucleotide sequence is provided in SEQ ID NO:331, and the encodedpolypeptide sequence is provided in SEQ ID NO:332.

Example 71 CTLA4 (CCC-P) WH WCH2 WCH3

This construct has a CTLA-4 binding region as described in Example 14.This binding region is attached to a wild type human IgG1 connectingregion (CCC-P) as described in Example 1. This connecting region isconnected to a wild type human IgG1 CH2 and CH3 region as described inExample 1. This construct has previously been referred to as CTLA-4 IgGWTH (CCC) WTCH2CH3, which has the same sequence as the above construct.The polynucleotide sequence is provided in SEQ ID NO:47, and the encodedpolypeptide sequence is provided in SEQ ID NO:78.

Example 72 FC2-2 scFv (SSS-S) H WCH2 WCH3

This construct has a FC2-2 (anti-CD16) single chain Fv made according tomethods described in Example 46. This binding region is connected to amutated human IgG1 connecting region where all of the cysteines and oneproline have been changed to serines (SSS-S) according to methodsdescribed in Example 5. This connecting region is connected to a wildtype human IgG1 CH2 and CH3 region as described in Example 1. Thepolynucleotide sequence is provided in SEQ ID NO:343, and the encodedpolypeptide sequence is provided in SEQ ID NO:344.

Example 73 FC2-2 scFv VHL11S (SSS-S) H WCH2 WCH3

This construct has a FC2-2 (anti-CD16) single chain Fv with a pointmutation at amino acid residue 11 in the heavy chain variable region,where the leucine has been changed to a serine as described in Example46. This binding region is connected to a mutated human IgG1 connectingregion where all of the cysteines and one proline have been changed toserines (SSS-S) according to methods described in Example 5. Thisconnecting region is connected to a wild type human IgG1 CH2 and CH3region as described in Example 1. The polynucleotide sequence isprovided in SEQ ID NO:345, and the encoded polypeptide sequence isprovided in SEQ ID NO:346.

Example 74 UCHL-1 scFv (SSS-S) H WCH2 WCH3

This construct has a UCHL-1 (anti-CD45RO) single chain Fv made accordingto methods described in Example 48. This binding region is connected toa mutated human IgG1 connecting region where all of the cysteines andone proline have been changed to serines (SSS-S) according to methodsdescribed in Example 5. This connecting region is connected to a wildtype human IgG1 CH2 and CH3 region as described in Example 1. Thepolynucleotide sequence is provided in SEQ ID NO:357, and the encodedpolypeptide sequence is provided in SEQ ID NO:358.

Example 75 UCHL-1 scFv VHL11S (SSS-S) H WCH2 WCH3

This construct has a UCHL-1 (anti-CD45RO) single chain Fv with a pointmutation at amino acid residue 11 in the heavy chain variable region,where the leucine has been changed to a serine as described in Example48. This binding region is connected to a mutated human IgG1 connectingregion where all of the cysteines and one proline have been changed toserines (SSS-S) according to methods described in Example 5. Thisconnecting region is connected to a wild type human IgG1 CH2 and CH3constant region as described in Example 1. The polynucleotide sequenceis provided in SEQ ID NO:359, and the encoded polypeptide sequence isprovided in SEQ ID NO:360.

Example 76 5B9 scFv (SSS-S) H WCH2 WCH3

This construct has a 5B9 (anti-CD137) single chain Fv made according tomethods described in Example 47. This binding region is connected to amutated human IgG1 connecting region where all of the cysteines and oneproline have been changed to serines (SSS-S) according to methodsdescribed in Example 5. This connecting region is connected to a wildtype human IgG1 CH2 and CH3 constant region as described in Example 1.This construct has previously been referred to as 5B9 scFv IgG MTH (SSS)WTCH2CH3, which has the same sequence as the above construct. Thepolynucleotide sequence is provided in SEQ ID NO:132, and the encodedpolypeptide sequence is provided in SEQ ID NO:133.

Example 77 5B9 scFv VHL11S (SSS-S) H WCH2 WCH3

This construct has a 5B9 (anti-CD137) single chain Fv with a pointmutation at amino acid residue 11 in the heavy chain variable region,where the leucine has been changed to a serine as described in Example47. This binding region is connected to a mutated human IgG1 connectingregion where all of the cysteines and one proline have been changed toserines (SSS-S) according to methods described in Example 5. Thisconnecting region is connected to a wild type human IgG1 CH2 and CH3constant region as described in Example 1. The polynucleotide sequenceis provided in SEQ ID NO:365, and the encoded polypeptide sequence isprovided in SEQ ID NO:366.

Example 78 2H7 scFv (SSS-S) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is connected to a wild type IgG1 CH2and CH3 constant region as described in Example 1. This construct haspreviously been referred to as 2H7-MHWTG1C, CytoxB-(MHWTG1C)—Ig,anti-CD20 scFv IgG MTH (SSS) WTCH2CH3, CytoxB-MHWTG1C, 2H7 scFv-humanIgG1 wild type hinge-CH2-CH3, and 2H7 scFv IgG MTH (SSS) WTCH2CH3, whichall have the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:57, and the encoded polypeptidesequence is provided in SEQ ID NO:58.

Example 79 2H7 scFv (SSS-S) H P238SCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This binding region is connected to a mutated human IgG1connecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.This hinge region is attached to a mutated human IgG1 CH2 region and awild type human IgG1 CH3 region. The P238S mutation, where a proline atresidue 238 was changed to a serine, was introduced according to methodsdescribed in Example 70. This construct has previously been referred toas 2H7 scFv IgG MTH (SSS) MTCH2WTCH3, anti-CD20 scFv IgG MTH (SSS)MTCH2CH3, and CytoxB-MHMG1C which all have the same sequence as theabove construct. The polynucleotide sequence is provided in SEQ IDNO:419, and the encoded polypeptide sequence is provided in SEQ IDNO:420.

Example 80 2H7 scFv IgAH WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a human IgAconnecting region and wild type human IgG1 CH2 and CH3 constant regionsas described in Example 5. This construct has previously been referredto as 2H7 scFv IgAH IgG WTCH2CH3, 2H7 scFv IgA hinge-IgG1 CH2-CH3, andCytoxB-IgAHWTHG1C, which all have the same sequence as the aboveconstruct. The polynucleotide sequence is provided in SEQ ID NO:59, andthe encoded polypeptide sequence is provided in SEQ ID NO:60.

Example 81 2H7 scFv IgAH WIgACH2 T4CH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a connectingregion from human IgA as described in Example 5. This connecting regionis attached to a human IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a truncation of 4 aminoacid residues prior to the 3′ stop codon as described in Example 13.This construct has previously been referred to as 2H7 scFv IgAH IgAT4,which has the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:70, and the encoded polypeptidesequence is provided in SEQ ID NO:71.

Example 82 2H7 scFv IgAH WIgACH2 WCH3+Jchain

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a wild typehuman IgA connecting region as described in Example 5. This connectingregion is attached to a wild type human IgA CH2 and CH3 constant regionaccording to methods described in Example 13. This constant region isattached to a J-chain region as described in Example 13. Thepolynucleotide sequence is provided in SEQ ID NO:61, and the encodedpolypeptide sequence is provided in SEQ ID NO:62.

Example 83 2H7 scFv (CCC-P) WH WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a wild typehuman IgG1 connecting region (CCC-P) as described in Example 1. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This construct haspreviously been referred to as 2H7 scFv Ig WTH (CCC) WTCH2CH3, 2H7 scFvIgG WTH WTCH2CH3, and 2H7 scFv-Ig, which both have the same sequence asthe above construct. The polynucleotide sequence is provided in SEQ IDNO:688, and the encoded polypeptide sequence is provided in SEQ IDNO:689.

Example 84 2H7 scFv (SSS-S) H WCH2 F405YCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to a wild type human IgG1CH2 and a mutated human IgG1 CH3 region. The F405Y mutation, where thephenylalanine at residue 405 has been changed to a tyrosine, wasintroduced according to methods described in Example 21. This constructhas previously been referred to as 2H7 scFv MTH WTCH2 MTCH3 Y405, whichhas the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:153, and the encoded polypeptidesequence is provided in SEQ ID NO:154.

Example 85 2H7 scFv (SSS-S) H WCH2 F405ACH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to a wild type human IgG1CH2 and a mutated human IgG1 CH3 region. The F405A mutation, where thephenylalanine at residue 405 has been changed to an alanine, wasintroduced according to methods described in Example 21. This constructhas previously been referred to as 2H7 scFv MTH WTCH2 MTCH3 A405, whichhas the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:155, and the encoded polypeptidesequence is provided in SEQ ID NO:156.

Example 86 2H7 scFv (SSS-S) H WCH2 Y407ACH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to a wild type human IgG1CH2 and a mutated human IgG1 CH3 region. The Y407A mutation, where thetyrosine at residue 407 has been changed to an alanine, was introducedaccording to methods described in Example 21. This construct haspreviously been referred to as scFv MTH WTCH2 MTCH3 A407, which has thesame sequence as the above construct. The polynucleotide sequence isprovided in SEQ ID NO:157, and the encoded polypeptide sequence isprovided in SEQ ID NO:158.

Example 87 2H7 scFv (SSS-S) HWCH2 F405A, Y407ACH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S), according to methods described inExample 5. This connecting region is attached to a wild type human IgG1CH2 and a mutated human IgG1 CH3 region. The F405A and Y407A mutation,where the phenylalanine at residue 405 has been changed to an alanineand the tyrosine at residue 407 has been changed to an alanine, wasintroduced according to methods described in Example 21. This constructhas previously been referred to as scFv MTH WTCH2 MTCH3 A405A407, whichhas the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:161, and the encoded polypeptidesequence is provided in SEQ ID NO:162.

Example 88 2H7 scFv (CSS-S) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a mutanthuman IgG1 connecting region, where the second and third cysteines havebeen changed to serines and the proline has been changed to serine(CSS-S), according to methods described in Example 13. This connectingregion is attached to wild type human IgG1 CH2 and CH3 constant regionsas described in Example 1. This construct has previously been referredto as 2H7 scFv MTH (CSS) WTCH2CH3, which has the same sequence as theabove construct. The polynucleotide sequence is provided in SEQ IDNO:134, and the encoded polypeptide sequence is provided in SEQ IDNO:135.

Example 89 2H7 scFv (SCS-S) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a mutanthuman IgG1 connecting region, where the first and third cysteines havebeen changed to serines and the proline has been changed to serine(SCS-S), according to methods described in Example 13. This connectingregion is attached to wild type human IgG1 CH2 and CH3 constant regionsas described in Example 1. This construct has previously been referredto as 2H7 scFv IgG MTH (SCS) WTCH2CH3, which has the same sequence asthe above construct. The polynucleotide sequence is provided in SEQ IDNO:136, and the encoded polypeptide sequence is provided in SEQ IDNO:137.

Example 90 2H7 scFv (SSC-P) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a mutanthuman IgG1 connecting region, where the first and second cysteines havebeen changed to serines (SSC-P), according to methods described inExample 13. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. This constructhas previously been referred to as 2H7 scFv MTH (SSC) WTCH2CH3, whichhas the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:138, and the encoded polypeptidesequence is provided in SEQ ID NO:139.

Example 91 2H7 scFv (CSC-S) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a mutanthuman IgG1 connecting region, where the second cysteine and the prolinehas been changed to serine (CSC-S), according to methods described inExample 32. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. This constructhas previously been referred to as 2H7 scFv MTH (CSC) WTCH2CH3, whichhas the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:165, and the encoded polypeptidesequence is provided in SEQ ID NO:166.

Example 92 2H7 scFv (CCS-P) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a mutanthuman IgG1 connecting region, where third cysteine has been changed to aserine (CCS-P), according to methods described in Example 22. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This construct haspreviously been referred to as 2H7 scFv MTH (CCS) WTCH2CH3, which hasthe same sequence as the above construct. The polynucleotide sequence isprovided in SEQ ID NO:167, and the encoded polypeptide sequence isprovided in SEQ ID NO:168.

Example 93 2H7 scFv (SCC-P) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a mutanthuman IgG1 connecting region, where first cysteine has been changed to aserine (SCC-P), according to methods described in Example 32. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This construct haspreviously been referred to as 2H7 scFv MTH (SCC) WTCH2CH3, which hasthe same sequence as the above construct. The polynucleotide sequence isprovided in SEQ ID NO:163, and the encoded polypeptide sequence isprovided in SEQ ID NO:164.

Example 94 2H7 scFv VH L11S (SSS-S) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine as described inExample 33. This binding region is connected to a mutated human IgG1connecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.This connecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This construct haspreviously been referred to as 2H7 scFv VH11SER IgG MTH (SSS) WTCH2CH3and 2H7 scFv VHSER11 WTH WTCH2CH3, which both have the same sequence asthe above construct. The polynucleotide sequence is provided in SEQ IDNO:369, and the encoded polypeptide sequence is provided in SEQ IDNO:370.

Example 95 2H7 scFv VH L11S (CSS-S) H WCH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine as described inExample 33. This binding region is attached to a mutant human IgG1connecting region, where the second and third cysteines have beenchanged to serines and the proline has been changed to serine (CSS-S),according to methods described in Example 13. This connecting region isattached to wild type human IgG1 CH2 and CH3 constant regions asdescribed in Example 1. The polynucleotide sequence is provided in SEQID NO:371, and the encoded polypeptide sequence is provided in SEQ IDNO:372.

Example 96 G28-1 scFv VH L11S (SCS-S) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a mutanthuman IgG1 connecting region, where the first and third cysteines havebeen changed to serines and the proline has been changed to serine(SCS-S), according to methods described in Example 13. This connectingregion is attached to wild type human IgG1 CH2 and CH3 constant regionsas described in Example 1. The polynucleotide sequence is provided inSEQ ID NO:373, and the encoded polypeptide sequence is provided in SEQID NO:374.

Example 97 G28-1 scFv V11 L11S(CCS-P) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a mutanthuman IgG1 connecting region, where third cysteine has been changed to aserine (CCS-P), according to methods described in Example 22. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. The polynucleotide sequenceis provided in SEQ ID NO:375, and the encoded polypeptide sequence isprovided in SEQ ID NO:376.

Example 98 G28-1 scFv VH L11S (SCC-P) H WCH2 WCH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a mutanthuman IgG1 connecting region, where first cysteine has been changed to aserine (SCC-P), according to methods described in Example 32. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. The polynucleotide sequenceis provided in SEQ ID NO:377, and the encoded polypeptide sequence isprovided in SEQ ID NO:378.

Example 99 G28-1 scFv VH L11S mIgE CH2 CH3 CH4

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a wilt typemouse IgE CH2 CH3 and CH4 region using methods described in Example 39.The polynucleotide sequence is provided in SEQ ID NO:379, and theencoded polypeptide sequence is provided in SEQ ID NO:380.

Example 100 G28-1 scFv V11 L11S mIgAH WIgACH2 T4CH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a connectingregion from mouse IgA as described in Example 39. This connecting regionis attached to a mouse IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a 4 amino acid truncationat residues as described in Example 39. The polynucleotide sequence isprovided in SEQ ID NO:381, and the encoded polypeptide sequence isprovided in SEQ ID NO:382.

Example 101 G28-1 scFv VH L11S hIgE CH2 CH3 CH4

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a wild typehuman IgE constant region containing CH2, CH3 and CH4 as described inExample 38. The polynucleotide sequence is provided in SEQ ID NO:383,and the encoded polypeptide sequence is provided in SEQ ID NO:384.

Example 102 G28-1 scFv VH L118 hIgAH WIgACH2 T4CH3

This construct has a G28-1 (anti-CD37) single chain Fv binding regionwith a point mutation at amino acid residue 11 in the heavy chainvariable region, where the leucine has been changed to a serine asdescribed in Example 35. This binding region is attached to a connectingregion from human IgA as described in Example 5. This connecting regionis attached to a human IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a truncation of 4 aminoacid residues prior to the 3′ stop codon as described in Example 13. Thepolynucleotide sequence is provided in SEQ ID NO:385, and the encodedpolypeptide sequence is provided in SEQ ID NO:386.

Example 103 HD37 scFv IgAH WCH2 WCH3

The HD37 scFv was cloned from the HD37 hybridoma using the Novagen -Igfamily primer sets, TOPO cloning and sequencing and sewing PCR assay.For the initial PCR reactions prior to sewing, the TOPO clone templatesHD37 VH C-1 and HD37 KVL B-9 were used at 1:100 with an amplificationprofile of 94 C 30 sec; 55 C, 30 sec; 72 C, 30 seconds for 25 cycles. Toprovide templates for the secondary SEWING PCR reactions, primaryreaction products were gel purified, QIAQUICK purified, and the eluatesdiluted 50 fold. One microliter each VL and VH overlapping templateswere added to PCR reactions with the following amplification profile: 94C, 60 sec; 55 C, 60 sec; 72 C, 60 sec; for 30 cycles. After two cycles,the machine was paused, and the flanking 5′VL and 3′VH primers wereadded to the reactions at 25 pmol each, and the PCR reactions resumed.PCR products were checked on a gel for the presence of an 750-800 byfragment, and the reactions products QIAQUICK purified and digested withthe appropriate restriction enzymes for insertion into pD18 Igexpression vectors.

PCR of VL domain with native leader peptide and part of glyser linker:

5′ primer: 5′-gttgttaagcttgccgccatggagacagacacactc (SEQ ID NO: 650)ctgctatgg-3′ 3′ primer: 5′gccacccgacccaccaccgcccgagccaccgccacct (SEQ IDNO: 651) ttgatttccagcttggtgcctcc-3′

PCR of VL domain without leader peptide (SalI site) and part of glyserlinker:

5′ primer: 5′-gttgttgtcgacattgtgctgacccaatctcca-3′ (SEQ ID NO: 652)3′ primer: 5′-gccacccgacccaccaccgcccgagccaccgccacc (SEQ ID NO: 651)tttgatttccagcttggtgcctcc-3′

PCR of VH domain with part of glyser linker and BclI site for fusion to-Ig tails.

5′ primer: 5′-tcgggcggtgggggtcgggtggcggcggatcgtca (SEQ ID NO: 653)caggttcagctgcagcagtctgg-3′ 3′ primer:5′-tcagtgctgatcagaggagacggtgactgaggttc (SEQ ID NO: 654) cttg-3′

This binding region is connected to a wild type human IgA connectingregion and wild type human IgG CH2 and CH3 constant regions as describedin Example 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. This constructhas previously been referred to as HD37 scFv-IgAHWTG1C andHD37-IgAHWTG1C, which both have the same sequence as the aboveconstruct. The polynucleotide sequence is provided in SEQ ID NO:397, andthe encoded polypeptide sequence is provided in SEQ ID NO:398.

Example 104 HD37 scFv (SSS-S) H WCH2 WCH3

This construct has a HD 37 single chain Fv as described in Example 103.This binding region is connected to a mutated human IgG1 connectingregion where all of the cysteines and one proline have been changed toserines (SSS-S) according to methods described in Example 5. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This construct haspreviously been referred to as HD37-MHWTG1C and HD37 scFv-IgMHWTG1C,which both have the same sequence as the above construct. Thepolynucleotide sequence is provided in SEQ ID NO:399, and the encodedpolypeptide sequence is provided in SEQ ID NO:400.

Example 105

HD37 scFv VH L11S (SSS-S) H WCH2 WCH3

This construct has a HD 37 single chain Fv with a mutation in the heavychain variable region at amino acid residue 11, where leucine has beenchanged to serine according to the methods described in Example 50. Thisbinding region is connected to a mutated human IgG1 connecting regionwhere all of the cysteines and one proline have been changed to serines(SSS-S) according to methods described in Example 5. This connectingregion is attached to wild type human IgG1 CH2 and CH3 constant regionsas described in Example 1. The polynucleotide sequence is provided inSEQ ID NO:401, and the encoded polypeptide sequence is provided in SEQID NO:402.

Example 106 L6 scFv IgAH WCH2 WCH3

The L6 scFv was cloned from the L6 hybridoma (1Hellstrom) using theanchor-tailing method described in the Tissue Antigens Paper from 1996.The PCR profile was 94 C, 1 min; 50 C, 2 min; 72 C, 2 min; for 35cycles. Once consensus sequence was obtained for VL and VH regions fromat least 4 TOPO clones, primers were ordered for PCR reactions prior toSEWING PCR reactions as follows: For the initial PCR reactions prior tosewing, the TOPO cloned templates L6VK and L6VH were used at 1:100 withan amplification profile of 94 C 30 sec; 55 C, 30 sec; 72 C, 30 secondsfor 25 cycles. To provide templates for the secondary SEWING PCRreactions, primary reaction products were gel purified, QIAQUICKpurified, and the eluates diluted 50 fold. One microliter each VL and VHoverlapping templates were added to PCR reactions with the followingamplification profile: 94 C, 60 sec; 55 C, 60 sec; 72 C, 60 sec; for 30cycles. After two cycles, the machine was paused, and the flanking 5′VLand 3′VH primers were added to the reactions at 25 pmol each, and thePCR reactions resumed. PCR products were checked on a gel for thepresence of an 750-800 by fragment, and the reactions products QIAQUICKpurified and digested with the appropriate restriction enzymes forinsertion into pD18 Ig expression vectors.

PCR of VL domain with native leader peptide and part of glyser linker:

L6VLHindIII: 5′-gttgttaagcttgccgccatggattttcaagtgcag (SEQ ID NO: 655)attttcagcttc-3′ L6VLLK3: 5′-gccacccgacccaccaccgcccgagccaccgccacc (SEQ IDNO: 656) agagagctcmcagctccagcug-3′

PCR of VL domain without leader peptide (SalI site) and part of glyserlinker:

5′ primer: 5′-gttgttgtcgacattgttctctcccagtctccagca (SEQ ID NO: 657)atcctgtctg-3′ 3′ primer: 5′-gccacccgacccaccaccgcccgagccaccgccacc (SEQ IDNO: 656) agagagctctttcagctccagcttggt-3′

PCR of VH domain with part of glyser linker and BclI site for fusion to-Ig tails.

5′: 5′-tcgggcggtggtgggtcgggtggcggcggatctctg (SEQ ID NO: 658)cagatccagttggtgcagtct-3′ 3′Bcl: 5′-tcagtgctgatcagaggagactgtgagagtggtgcc(SEQ ID NO: 659) ttg-3′

This binding region is connected to a human IgA connecting region andwild type human IgG1 CH2 and CH3 constant regions as described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. This constructhas previously been referred to as L6 scFv-IgAHWTG1C, which HAS the samesequence as the above construct. The polynucleotide sequence is providedin SEQ ID NO:413, and the encoded polypeptide sequence is provided inSEQ ID NO:414.

Example 107 L6 scFv VHL11S (SSS-S) H WCH2 WCH3

This construct has a L6 single chain Fv with a mutation in the heavychain variable region at amino acid residue 11, where leucine has beenchanged to serine according to the methods described in Example 49. Thisbinding region is connected to a mutated human IgG1 connecting regionwhere all of the cysteines and one proline have been changed to serines(SSS-S) according to methods described in Example 5. This connectingregion is attached to wild type human IgG1 CH2 and CH3 constant regionsas described in Example 1. The polynucleotide sequence is provided inSEQ ID NO:415, and the encoded polypeptide sequence is provided in SEQID NO:416.

Example 108 2H7 scFv-Llama IgG1

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a llama IgG1hinge, CH2 and CH3 regions according to the methods described in Example10. The polynucleotide sequence is provided in SEQ ID NO:21, and theencoded polypeptide sequence is provided in SEQ ID NO:22.

Example 109 2H7 scFv-Llama IgG2

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a llama IgG2hinge, CH2 and CH3 regions according to the methods described in Example10. The polynucleotide sequence is provided in SEQ ID NO:23, and theencoded polypeptide sequence is provided in SEQ ID NO:24.

Example 110 2H7 scFv-Llama IgG3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a llama IgG3hinge, CH2 and CH3 regions according to the methods described in Example10. The polynucleotide sequence is provided in SEQ ID NO:25, and theencoded polypeptide sequence is provided in SEQ ID NO:26.

Example 111 CD16 Low (ED)(SSS-S) H P238SCH2 WCH3

This construct has the extra cellular, CD16 low affinity allele bindingdomain as described in Example 43. This binding region is connected to amutated human IgG1 connecting region where all of the cysteines and oneproline have been changed to serines (SSS-S) according to methodsdescribed in Example 5. This hinge region is attached to a mutated humanIgG1 CH2 region and a wild type human IgG1 CH3 region. The P238Smutation, where a proline at residue 238 was changed to a serine, wasintroduced according to methods described in Example 70. Thepolynucleotide sequence is provided in SEQ ID NO:421, and the encodedpolypeptide sequence is provided in SEQ ID NO:422.

Example 112 CD16-9 High (ED)(SSS-S) H P238SCH2 WCH3

This construct has the extra cellular, CD16 high affinity allele bindingdomain as described in Example 43. This binding region is connected to amutated human IgG1 connecting region where all of the cysteines and oneproline have been changed to serines (SSS-S) according to methodsdescribed in Example 5. This hinge region is attached to a mutated humanIgG1 CH2 region and a wild type human IgG1 CH3 region. The P238Smutation, where a proline at residue 238 was changed to a serine, wasintroduced according to methods described in Example 70. Thepolynucleotide sequence is provided in SEQ ID NO:425, and the encodedpolypeptide sequence is provided in SEQ ID NO:426.

Example 113 2e12 scFv (SSS-S)H P238SCH2 WCH3-hCD80TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to a mutated human IgG1CH2 region and a wild type human IgG1 CH3 region. The P238S mutation,where a proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region is attachedto a human CD80 transmembrane and cytoplasmic tail region (hCD80 TM/CT).The hCD80 TM/CT was cloned using random primed cDNA derived from theBJAB cell line according to methods described in Example 12. This TM/CTregion was attached to a Ig CH3 region with an open reading frame (ORF).Open reading frame versions of scFvIg constructs of interest werecreated by replacement of the soluble versions of each -Ig tail with ORF(open reading frame versions) of these tails. PCR primers were designedfor the existing clones of soluble -Ig tails which delete the stop codonand add one or more restriction sites to the 3′ end of the new -Igcassettes. The desired transmembrane and cytoplasmic tail sequences canthen be subcloned downstream of these new -Ig cassettes. Each constructutilized the existing available 5′ BCLI oligonucleotide used inamplifying the soluble version of the tails for the PCR reactions. The3′ oligonucleotides replace the stop codon with out of frame restrictionsites fused to the coding region for protein domains involved inregulation of apoptosis.

The PCR amplifications were carried out with 25 pmol of each primer,standard PCR reagents, and varying volumes of either cloned domains orcDNA obtained from PBMC, spleen, or thymus RNA. The reactions used acycling profile of 94 C, 60sec; 55 C, 60 sec; 72 C, 2 min, for 35cycles. The primers for the IgG ORF are listed below.

5′ primer: 5′-gttgtagatcaggagcccaaatcttotgacaaaac (SEQ ID NO: 660)tcacacatctccaccgtccccagcacctgaactcctggg ggaccgtcagtcttcc-3′ 3′ primer:5′-gttgttttcgaaggatccgctttacccgggagcag (SEQ ID NO: 661)ggagaggctcttctgcgtgtagtg-3′

The polynucleotide sequence is provided in SEQ ID NO:437, and theencoded polypeptide sequence is provided in SEQ ID NO:438.

Example 114 10A8 scFv (SSS-S)H P238SCH2 WCH3-hCD80TM/CT

This construct has a 10A8 (anti-CD2152) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. This CH3 region isattached to a hCD80 TM/CT according to methods described in Example 113.The polynucleotide sequence is provided in SEQ ID NO:439, and theencoded polypeptide sequence is provided in SEQ ID NO:440.

Example 115 40.2.220 scFv (SSS-S)H P238SCH2 WCH3-hCD80TM/CT

This construct has a 40.2.220 (anti-CD40) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. This CH3 region isattached to a hCD80 TM/CT according to methods described in Example 113.

Example 116 2H7 scFv (SSS-S)H P238SCH2 WCH3-hCD80TM/CT

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S), according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. This CH3 region isattached to a hCD80 TM/CT according to methods described in Example 113.This construct has previously been referred to as: The polynucleotidesequence is provided in SEQ ID NO:441, and the encoded polypeptidesequence is provided in SEQ ID NO:442.

Example 117 G19-4 scFv (SSS-S)H P238SCH2 WCH3-hCD80TM/CT

This construct has a G19 (anti-CD3) single chain Fv binding regiondescribed in Example 29. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. This CH3 region isattached to a hCD80 TM/CT according to methods described in Example 113.The polynucleotide sequence is provided in SEQ ID NO:443, and theencoded polypeptide sequence is provided in SEQ ID NO:444.

Example 118 2E12 scFv (SSS-S)H WCH2 WCH3-hCD80TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. This CH3 regionis attached to a hCD80 TM/CT according to methods described in Example113. This construct has previously been referred to as 2e12 scFv IgG WTHWHTCH3CH2-CD80, which has the same sequence as the above construct. Thepolynucleotide sequence is provided in SEQ ID NO:126, and the encodedpolypeptide sequence is provided in SEQ ID NO:127.

Example 119 2E12 scFv IgAH IgACH2 T4CH3-hCD80TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is attached to a connectingregion from human IgA as described in Example 5. This connecting regionis attached to a human IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a truncation of 4 aminoacid residues prior to the 3′ stop codon as described in Example 13.This CH3 region is attached to a hCD80 TM/CT according to methodsdescribed in Example 113. The specific primers used to create an IgA ORFare listed below.

5′primer: 5′-gttgttgatcagccagttccctcaactccacctac (SEQ ID NO: 662) c-3′3′primer: 5′-gttgttttcgaaggatccgcgtccacctccgccatg (SEQ ID NO: 663)acaacaga

The polynucleotide sequence is provided in SEQ ID NO:445, and theencoded polypeptide sequence is provided in SEQ ID NO:446.

Example 120 2E12 scFv IgE CH2CH3CH4-hCD80TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is attached to a human IgEconstant region containing CH2, CH3 and CH4 as described in Example 38.This CH4 region is attached to a hCD80 TM/CT essentially according tomethods described in Example 113. The specific primers used to create anIgE ORF are listed below.

5′ primer: 5′-gttgttgatcacgtctgctccagggacttcacc-3′ (SEQ ID NO: 664)3′ primer: 5′-gttgttttcgaaggatccgctttaccagatttacag (SEQ ID NO: 665)acaccgctcgctg-3′

The polynucleotide sequence is provided in SEQ ID NO:183, and theencoded polypeptide sequence is provided in SEQ ID NO:184.

Example 121 2E12 scFv (SSS-S)H P238SCH2 WCH3-mFADD-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region is attachedto a mouse FADD transmembrane ans cytoplasmic tail region (mFADD TM/CT).This region is cloned using essentially the same methods described inExample 113. The domain was PCR amplified from randomly primed cDNA frommouse spleen RNA. The specific primers are listed below.

5′ primer: 5′-gttgtggatccttcgaacccattcctggtgctgctg (SEQ ID NO: 666)cactcgctg-3′ 3′ primer: 5′-gttgttatcgatctcgagtcagggtgtttctgagga (SEQ IDNO: 667) agacacagt-3′

The specific primers used to create a mouse IgG ORF are listed below.

5′primer: 5′-gttgtagatctggagcccagagggcccacaatcaag (SEQ ID NO: 668)ccctctcctccaagcaaaagccca-3′ 3′primer:5′-gttgttttcgaaggatccgctttacccggagtccgg (SEQ ID NO: 669) gagaag-3′

The polynucleotide sequence is provided in SEQ ID NO:447, and theencoded polypeptide sequence is provided in SEQ ID NO:448.

Example 122 2E12 scFv (SSS-S)H WCH2 WCH3-mFADD-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionwas attached to a mFADD TM/TM region according to methods described inExample 113 and 121. The polynucleotide sequence is provided in SEQ IDNO:449, and the encoded polypeptide sequence is provided in SEQ IDNO:450.

Example 123 2E12 scFv (SSS-S)H WCH2 WCH3-mcasp3-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionis attached to a mouse casp3 transmembrane and ctyoplasmic tail regionaccording to methods described in Examples 113 and 121. The specificprimers used to isolate the mcasp3 TM/CT region are listed below:

5′ primer: 5′-gttgttggatccttcgaacatggagaacaacaaaac (SEQ ID NO: 670)ctcagtggattca-3′ 3′ primer: 5′-gttgttatcgatctcgagctagtgataaaagtacag (SEQID NO: 671) ttctttcgt-3′

The polynucleotide sequence is provided in SEQ ID NO:453, and theencoded polypeptide sequence is provided in SEQ ID NO:454.

Example 124 2E12 scFv (SSS-S)H P238SCH2 WCH3-mcasp3-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region isconnected to a mcasp3 TM/CT region according to methods described inExamples 113, 121 and 123. The polynucleotide sequence is provided inSEQ ID NO:455, and the encoded polypeptide sequence is provided in SEQID NO:456.

Example 125 2E12 scFv (SSS-S)H WCH2 WCH3-mcasp8-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionis attached to a mouse casp8 transmemebrane and cytoplasmic tail region(mcasp8 TM/CT) essentially according to methods described in Example 113and 121. The specific primers used to clone the mcasp8 TM/CT region arelisted below.

5′ primer: 5′-gttgtttcgaacatggatttccagagttgtctttat (SEQ ID NO: 672)gctattgctg-3′ 3′ primer: 5′-gttgttatcgatctcgagtcattagggagggaagaa (SEQ IDNO: 673) gagcttcttcg-3′

The polynucleotide sequence is provided in SEQ ID NO:459, and theencoded polypeptide sequence is provided in SEQ ID NO:460.

Example 126 2E12 scFv (SSS-S)H P238SCH2 WCH3-mcasp8-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region wasattached to a mcasp8 TM/CT region according to methods described inExamples 113, 121 and 125. The polynucleotide sequence is provided inSEQ ID NO:461, and the encoded polypeptide sequence is provided in SEQID NO:462.

Example 127 2E12 scFv (SSS-S)H WCH2 WCH3-Hcasp3-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionwas attached to a human casp3 transmembrane and cytoplasmic tail region(hcasp3 TM/CT) essentially according to methods describe in Examples113. The specific primers used to clone the hcasp3 TM/CT region arelisted below.

5′ Primer: 5′-gttgtggatccttcgaacatggagaacactgaaaac (SEQ ID NO: 674)tcagtggat-3′ 3′ Primer: 5′-gttatcgatctcgagtagtgataaaaatagagttct (SEQ IDNO: 675) tttgtgag-3′

The polynucleotide sequence is provided in SEQ ID NO:465, and theencoded polypeptide sequence is provided in SEQ ID NO:466.

Example 128 2E12 scFv (SSS-S)H P238SCH2 WCH3-hcasp3-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region is attachedto a hcasp3 TM/CT region according to methods described in Examples 113and 127. The polynucleotide sequence is provided in SEQ ID NO:467, andthe encoded polypeptide sequence is provided in SEQ ID NO:468.

Example 129 2E12 scFv (SSS-S)H WCH2 WCH3-hcasp8-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionis attached to a human casp8 transmembrane and cytoplasmic tail region(hcasp8 TM/CT) according to methods described in Example 133. Thespecific primers used to clone the hcasp8 TM/CT region are listed below.

5′ Primer: (SEQ ID NO: 676)5′-gttgtggatccttcgaacatggacttcagcagaaatctttatg at-3′ 3′ Primer: (SEQ IDNO: 677) 5′-gttgttatcgatgcatgctcaatcagaagggaagacaagttttttt ct-3′

The polynucleotide sequence is provided in SEQ ID NO:473, and theencoded polypeptide sequence is provided in SEQ ID NO:474.

Example 130 2E12 scFv (SSS-S)H P238SCH2 WCH3-hcasp8-TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region is attachedto a hcasp8 TM/CT according to methods described in Examples 113 and129. The polynucleotide sequence is provided in SEQ ID NO:475, and theencoded polypeptide sequence is provided in SEQ ID NO:476.

Example 131 1D8 scFv (SSS-S)H P238SCH2 WCH3-HCD80TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region is attachedto a hCD80 TM/CT region according to methods described in Example 113.The polynucleotide sequence is provided in SEQ ID NO:108, and theencoded polypeptide sequence is provided in SEQ ID NO:109.

Example 132 1D8 scFv (SSS-S)H WCH2 WCH3-hCD80TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionis attached to a hCD80 TM/CT region according to methods described inExample 113. This construct has previously been referred to as 1D8 scFvIgG WTH WTCH2CH3-CD80, which has the same sequence as the aboveconstruct. The polynucleotide sequence is provided in SEQ ID NO:481, andthe encoded polypeptide sequence is provided in SEQ ID NO:482.

Example 133 1D8 scFv-mIgAH WIgA CH2 T4CH3-hCD80TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is attached to a connectingregion from human IgA as described in Example 5. This connecting regionis attached to a mouse IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a truncation of 4 aminoacid residues prior to the 3′ stop codon as described in Example 39. TheCH3 region can be attached to a hCD80 TM/CT region according to methodsdescribed in Examples 113 using primers that create an IgA ORF (SEQ IDNOs: 483 and 484).

Example 134 1D8 scFv IgE CH2CH3CH4-HCD80TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is attached to a human IgEconstant region containing CH2, CH3 and CH4 as described in Example 38.The CH4 region is attached to a hCD80 TM/CT according to methodsdescribed in Examples 113 and 120. The polynucleotide sequence isprovided in SEQ ID NO:175, and the encoded polypeptide sequence isprovided in SEQ ID NO:176.

Example 135 1D8 scFv (SSS-S)H P238SCH2 WCH3-mFADD-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region wasattached to a mFADD TM/TM region according to methods described inExample 113 and 121. The polynucleotide sequence is provided in SEQ IDNO:487, and the encoded polypeptide sequence is provided in SEQ IDNO:488.

Example 136 1D8 scFv (SSS-S)H WCH2 WCH3-mFADD-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionwas attached to a mFADD TM/TM region according to methods described inExample 113 and 121. The polynucleotide sequence is provided in SEQ IDNO:485, and the encoded polypeptide sequence is provided in SEQ IDNO:486.

Example 137 1D8 scFv (SSS-S)H WCH2 WCH3-mcasp3-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionwas attached to a mcasp3 TM/TM region according to methods described inExample 113, 121 and 123. The polynucleotide sequence is provided in SEQID NO:489, and the encoded polypeptide sequence is provided in SEQ IDNO:490.

Example 138 1D8 scFv (SSS-S)H P238SCH2 WCH3-mcasp3-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region wasattached to a mcasp3 TM/TM region according to methods described inExample 113, 121 and 123. The polynucleotide sequence is provided in SEQID NO:491, and the encoded polypeptide sequence is provided in SEQ IDNO:492.

Example 139 1D8 scFv (SSS-S)H WCH2 WCH3-mcasp8-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionwas attached to a mcasp8 TM/TM region according to methods described inExample 113, 121 and 125. The polynucleotide sequence is provided in SEQID NO:493, and the encoded polypeptide sequence is provided in SEQ IDNO:494.

Example 140 1D8 scFv (SSS-S)H P238SCH2 WCH3-mcasp8-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region wasattached to a mcasp8 TM/TM region according to methods described inExample 113, 121 and 125. The polynucleotide sequence is provided in SEQID NO:495, and the encoded polypeptide sequence is provided in SEQ IDNO:496.

Example 141 1D8 scFv (SSS-S)H WCH2 WCH3-hcasp3-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionis attached to a hcasp3 TM/CT according to methods described in Examples113 and 127. The polynucleotide sequence is provided in SEQ ID NO:497,and the encoded polypeptide sequence is provided in SEQ ID NO:498.

Example 142 1D8 scFv (SSS-S)H P238SCH2 WCH3-hcasp3-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region is attachedto a hcasp3 TM/CT according to methods described in Examples 113 and127. The polynucleotide sequence is provided in SEQ ID NO:499, and theencoded polypeptide sequence is provided in SEQ ID NO:500.

Example 143 1D8 scFv (SSS-S)H WCH2 WCH3-hcasp8-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This connecting region is attached to wild type human IgG1CH2 and CH3 constant regions as described in Example 1. The CH3 regionis attached to a hcasp8 TM/CT according to methods described in Examples113 and 129. The polynucleotide sequence is provided in SEQ ID NO:501,and the encoded polypeptide sequence is provided in SEQ ID NO:502.

Example 144 1D8 scFv (SSS-s)H P238SCH2 WCH3-hcasp8-TM/CT

This construct has a 1D8 (anti-4-1BB) single chain Fv binding regiondescribed in Example 25. This binding region is connected to a mutatedhuman IgG1 connecting region where all of the cysteines and one prolinehave been changed to serines (SSS-S) according to methods described inExample 5. This hinge region is attached to a mutated human IgG1 CH2region and a wild type human IgG1 CH3 region. The P238S mutation, wherea proline at residue 238 was changed to a serine, was introducedaccording to methods described in Example 70. The CH3 region is attachedto a hcasp8 TM/CT according to methods described in Examples 113 and129. The polynucleotide sequence is provided in SEQ ID NO:503, and theencoded polypeptide sequence is provided in SEQ ID NO:504.

Example 145 L6 scFv (SSS-S) H WCH2 WCH3

This construct has a L6 scFv binding domain as described in Example 105.This binding region is connected to a mutated human IgG1 connectingregion where all of the cysteines and one proline have been changed toserines (SSS-S) according to methods described in Example 5. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This construct haspreviously been referred to as L6 scFv-IgMHWTG1C, which has the samesequence as the above construct. The polynucleotide sequence is providedin SEQ ID NO:415, and the encoded polypeptide sequence is provided inSEQ ID NO:416.

Example 146 2H7 scFv CD154 (L2)

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region has been attached to CD154extracellular domain according to methods described in Example 4. Thepolynucleotide sequence is provided in SEQ ID NO:690, and the encodedpolypeptide sequence is provided in SEQ ID NO:691.

Example 147 2H7 scFv CD154 (S4)

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region has been attached to CD154extracellular domain according to methods described in Example 4, suchthat methods of attachment resulted in a truncated version compared tothe construct describe in Example 146. The polynucleotide sequence isprovided in SEQ ID NO:692, and the encoded polypeptide sequence isprovided in SEQ ID NO:693.

Example 148 CTLA4 IgAH IgACH2CH3

This construct has the extra cellular CTLA-4 binding region as describedin Example 14. This binding region is attached to a wild type human IgAconnecting region as described in Example 5. This connecting region isattached to a wild type human IgA CH2 and CH3 constant region accordingto methods described in Example 13. This constant region is attached toa J-chain region as described in Example 13. This construct haspreviously been referred to as CTLA-4 IgAH IgACH2CH3, which has the samesequence as the above construct. The polynucleotide sequence is providedin SEQ ID NO:505, and the encoded polypeptide sequence is provided inSEQ ID NO:506.

Example 149 CTLA4 IgAH IgACH2 T4-CH3

This construct has the extra cellular CTLA-4 binding region as describedin Example 14. This binding region is attached to a connecting regionfrom human IgA as described in Example 5. This connecting region isattached to a human IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a truncation of 4 aminoacid residues prior to the 3′ stop codon as described in Example 13.This construct has previously been referred to as CTLA-4 IgAH IgA-T4,which has the same sequence as the above construct. The polynucleotidesequence is provided in SEQ ID NO:507, and the encoded polypeptidesequence is provided in SEQ ID NO:508.

Example 150 2H7 scFv IgAH IgACH2CH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a wild typehuman IgA connecting region as described in Example 5. This connectingregion is attached to a wild type human IgA CH2 and CH3 constant regionaccording to methods described in Example 13. The polynucleotidesequence is provided in SEQ ID NO:61, and the encoded polypeptidesequence is provided in SEQ ID NO:62.

Example 151 2H7 scFv IgAH IgAHCH2 T18CH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a connectingregion from human IgA as described in Example 5. This connecting regionis attached to a human IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a truncation of 18 aminoacid residues prior to the 3′ stop codon as described in Example 13. Thepolynucleotide sequence is provided in SEQ ID NO:509, and the encodedpolypeptide sequence is provided in SEQ ID NO:510.

Example 152 2H7-40.2.220 scFv (SSS-S) H WCH2 WCH3

A bispecific fusion protein was constructed between 2H7 scFv (anti-CD20)and 40.2.220 (anti-CD40), which both target B cell receptors. The 2H7scFv hIgG1 (SSS-S)H WCH2 WCH3 construct in the expression vector pD18was passaged through dam⁻ bacteria in order to permit cleavage at theBclI site. Cleaved plasmid was treated with alkaline phosphatase priorto ligation to a BclI cut linker-CD40 scFv fragment. This fragment wassynthesized from the existing 40.2.220 scFv by successive PCR reactionswith overlapping primers. The linker attached is a previously patented(BMS patent issued) helical type linker with a high number of lysine andglutamic acid residues. The scFv for CD40 was PCR amplified without theleader peptide as a SalI-BclI fragment, but with the hinge type linkersubstituted at the amino terminus as a BclI-SalI fragment, to mediateinsertion of the linker-scFv cassette as a BclI fragment between thescFv and -Ig tail included in an existing scFvIg construct for CD20 (2H7scFv hIgG1 constructs). The 3′ end was similar to the other scFv VHmolecules with an out of frame BclI site fused to the VTVSS typesequence at the end of the VH domain.

PCR oligos:

40.2.220 scFv:

5′ primer--40.2.220S5: (SEQ ID NO: 678)5′-gttgttgtcgacattgttctgactcagtctccagccaccctgtc-3′3′ primer--40.2.220Bc13: (SEQ ID NO: 679)5′-gttgttgatcagagacagtgaccagtgtcccttgg-3′Linker Primers:BclI-SalI fragment created by annealing complementary oligonucleotides.This fragment is then ligated into BclI digested vector along with aSalI-BclI scFv to create the (linker-scFv) BclI fragment desired forshuttling.

5′ primer: (SEQ ID NO: 560)5′-gatcaatccaactctgaagaagcaaagaaagaggaggccaaaaaggaggaagccaagaaatctaacagcg-3′ 3′ primer: (SEQ ID NO: 563)5′-tcgacgctgttagatttcttggcttcctcctttttggcctcctctttctttgcttcttcagagttggatt-3′

This BclI fragment was then ligated downstream of the 2H7 scFv inpD18-Ig. Transformants were screened for the presence of a 2.4 kbHindIII-Xba insert and positive clones sequenced prior to furtherstudies. COS cell transient transfections were performed with thisconstruct and culture supernatants screened for the presence of proteinof the predicted size and for binding to CD20 and to CD40 transfectedCHO cells.

New bispecific constructs can be created by designing hinge type linkerswhich incorporate one or preferably two restriction sites at either endof the linker, facilitating asymmetric digests and transfer of(linker-scFv) or (scFv-linker) cassettes between different constructs.These constructs will also incorporate the VH L11S and other V regionsubstitutions, which presumably facilitate proper folding and result inincreased expression of the molecules in which they are inserted.

This binding region is connected to a mutated human IgG1 connectingregion where all of the cysteines and one proline have been changed toserines (SSS-S) according to methods described in Example 5. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This construct haspreviously been referred to as anti-CD20-anti-CD40 scFv IgG MTH (SSS)MTCH2WTCH3, which has the same sequence as the above construct. Thepolynucleotide sequence is provided in SEQ ID NO:114, and the encodedpolypeptide sequence is provided in SEQ ID NO:115.

Example 153 2H7 scFv IgAH IgACH2 T4-CH3-hCD80 TM/CT

This construct has a 2H7 (anti-CD20) single chain Fv binding region asdescribed in Example 1. This binding region is attached to a connectingregion from human IgA as described in Example 5. This connecting regionis attached to a human IgA constant region consisting of a wild type CH2region and a mutated CH3 region where there is a truncation of 4 aminoacid residues prior to the 3′ stop codon as described in Example 13.This CH3 region is attached to a hCD80 TM/CT according to the methodsdescribed in Examples 113 and 119. This construct has previously beenreferred to as 2H7 scFv IgA hinge IgA-T4-CD80 and 2H7 scFv IgAHIgA-T4-CD80, which both have the same sequence as the above construct.The polynucleotide sequence is provided in SEQ ID NOs:70 and 29, and theencoded polypeptide sequence is provided in SEQ ID NOs:71 and 30.

Example 154 G19-4 scFv (CCC-P) WH WCH2 WCH3-hCD80 TM/CT

This construct has a G19 (anti-CD3) single chain Fv binding regiondescribed in Example 29. This binding region is attached to a wild typehuman IgG1 connecting region (CCC-P) as described in Example 1. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This CH3 region is attachedto a hCD80 TM/CT according to the methods described in Examples 113. Thepolynucleotide sequence is provided in SEQ ID NOs:112 and 29, and theencoded polypeptide sequence is provided in SEQ ID NOs:113 and 30.

Example 155 2E12 scFv (CCC-P) WH WCH2 WCH3-HCD80 TM/CT

This construct has a 2e12 (anti-CD28) single chain Fv binding regiondescribed in Example 12. This binding region is attached to a wild typehuman IgG1 connecting region (CCC-P) as described in Example 1. Thisconnecting region is attached to wild type human IgG1 CH2 and CH3constant regions as described in Example 1. This CH3 region is attachedto a hCD80 TM/CT according to the methods described in Examples 113. Thepolynucleotide sequence is provided in SEQ ID NO:126, and the encodedpolypeptide sequence is provided in SEQ ID NO:127.

Example 156 2H7 VHL11S scFv (SSS-S) IgECH3CH4

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine as described inExample 33. This binding region is connected to a mutated human IgG1connecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.This connecting region is attached to human IgE CH3 and CH4 constantregion. This truncated constant region was created according to themethods described in Example 38. The polynucleotide sequence is providedin SEQ ID NO:227, and the encoded polypeptide sequence is provided inSEQ ID NO:228.

Example 157 IgD Hinge

An alternative hinge region can be isolated from human IgDimmunoglobulin hinge region by using PCR assay to isolate the desiredregion. The PCR reaction is the same used in Example 1. This hinge wastruncated by 6 amino acid residues at the 3′ end. The primers used inthis PCR reaction are listed below.

5″ Primer: (SEQ ID NO: 534) 5′-GTGGATCCAGGTTCGAAGTCTCGAAAGGCACAGGCC-3′3′ primer: (SEQ ID NO: 517) 5′-GTTGTCGACTGCACGGGTCTTTGTCTCTCTCTCTTC-3′

The polynucleotide sequence is provided in SEQ ID NO:237, and theencoded polypeptide sequence is provided in SEQ ID NO:238.

Example 158 hCD28 TM/CT

For some of the cell surface ORF constructs, the transmembrane domain ofCD80 was substituted with the transmembrane domain of human CD28 becauseit forms a dimer on the cell surface rather than a monomer as the CD80does. Several of the molecules which drive the apoptotic program requireoligomerization/trimerization to form a signaling complex; therefore, itis important to be able to control initiation of signaling bycontrolling the degree of oligomerization of these recombinant receptorson the cell surface.

The primers used in PCR amplification of the CD28 tail are given below:

5′ Primer: (SEQ ID NO: 518)5′-gttgtggatccttcgaaccccttttgggtgctggtggtggttggtg ga-3′ 3′ primer: (SEQID NO: 519) 5′-gttgttatcgatctcgagtcaggagcgataggctgcgaagtc-3′

Example 159 2H7 scFv VH L11S (SSS-S) H K322L CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine. as described inExample 33. This binding region is connected to a mutated human IgGconnecting region where all of the cysteines and one proline have beenchanged to serines (SSS-S) according to methods described in Example 5.The connecting region is attached to a mutated IgG CH2 region and a wildtype IgG CH3 region. The K322L mutation in the CH2 region is at aresidue 322, where a Lysine has been changed to a leucine usingoverlapping PCR described in Example 56, but with different primers forthe first PCR reaction, which are listed below.

5′ primer: (SEQ ID NO: 635)5′ttcctcttccccccaaaacccaaggacaccctcatgatctcccgga accctgaggtcac-3′3′ primer: (SEQ ID NO: 621) 5′-ggacagtgggagtggcacc-3′

PCR product was cloned into TOPO vector and sequenced. Thepolynucleotide sequence is provided in SEQ ID NO:265, and the encodedpolypeptide sequence is provided in SEQ ID NO:266.

Example 160 2H7 scFv VH L11S(CSS-S) H K322L CH2 WCH3

This construct has a 2H7 (anti-CD20) single chain Fv binding region witha point mutation at amino acid residue 11 in the heavy chain variableregion, where the leucine has been changed to a serine, as described inExample 33. This binding region is attached to a mutant IgG connectingregion, where the second and third cysteines have been changed toserines and the proline has been changed to serine (CSS-S), according tomethods described in Example 13. The connecting region is attached to amutated IgG CH2 region and a wild type IgG CH3 region. The K322Lmutation in the CH2 region is at a residue 322, where a Lysine has beenchanged to a leucine using overlapping PCR described in Example 56,using primers from Example 159 in the first PCR reaction and primersfrom Example 57 for the second PCR reaction.

PCR products were cloned into the TOPO vector and sequenced. Thepolynucleotide sequence is provided in SEQ ID NO:261, and the encodedpolypeptide sequence is provided in SEQ ID NO:262.

Example 161 In Vivo B Cell Depletion by 2H7 scFv (SSS-S) H WCH2 WCH3 and2H7 scFv (SSS-S) H P238SCH2 WCH3

This Example describes experiments relating to ADCC effector mechanismsin the depletion of B cells through comparison of the activities of ananti-CD20 construct (2H7 scFv (SSS-S) H WCH2 WCH3) and a anti-CD20construct with a proline to serine amino acid substitution in the CH2domain (2H7 scFv (SSS-S) H P238SCH2 WCH3). FIG. 72 shows the results ofan experiment on apoptosis induction by these constructs. The resultsindicated that both molecules bind CD20 and induce apoptosis that ismediated by activation of caspase 3.

FIG. 73 illustrates that anti-CD20 constructs mediate CDC activitytowards CD20 positive target cells. A 2H7 scFv (SSS-S) H WCH2 WCH3construct, a 2H7 scFv (SSS-S) H P238SCH2 WCH3 construct, or Rituximabwere incubated at increasing concentrations with 10⁴ Bjab Target Cellsand a 1:10 dilution of rabbit complement (PelFreez) in a volume of 100microliters for sixty minutes. Aliquots were stained with trypan blue(Invitrogen), and counted using a hemacytometer to determine thepercentage of the cell population killed during treatment. Negativecontrols with cells and only one reagent were also included. FIG. 74illustrates that the 2H7 scFv (SSS-S) H WCH2 WCH3 construct waseffective in ADCC with peripheral blood mononuclear cells. ADCC activityof 2H7 scFv (SSS-S) H WCH2 WCH3 or Rituximab was measured in vitroagainst BJAB B lymphoma cell line as the target cells, and using freshhuman PBMC as effector cells. Effector to target ratios were varied asfollows: 100:1, 50:1, and 25:1, with the number of BJAB cells per wellremaining constant but varying the number of PBMC. BJAB cells werelabeled for 2 hours with ⁵¹Cr and aliquoted at a cell density of 5×10⁴cells/well to each well of flat-bottom 96 well plates. Purified fusionproteins or Rituximab were added at a concentration of 10 μg/ml, andPBMC were added at 1.25×10⁶ cells/well (25:1), 2.5×10⁶ cells/well(50:1), or 5×10⁶ cells/well (100:1), in a final volume of 200 μl.Natural killing was measured at each effector:target ratio by omissionof the construct or MAb. Spontaneous release was measured withoutaddition of PBMC or fusion protein, and maximal release was measured bythe addition of detergent (1% NP-40) to the appropriate wells. Reactionswere incubated for 5 hours, and 100 μl culture supernatant harvested toa Lumaplate (Packard Instruments) and allowed to dry overnight prior tocounting cpm released on a Packard Top Count NXT MicroplateScintillation Counter. FIG. 75 illustrates that the 2H7 scFv (SSS-S) HWCH2 WCH3 construct bound soluble CD16 fusion protein (constructed fromeither high or low affinity alleles (158V/F)), while the 2H7 scFv(SSS-S) H P238SCH2 WCH3 construct did not show detectable binding. CD20CHO cells (106) were incubated with saturating amounts of 2H7 scFv(SSS-S) H WCH2 WCH3 or 2H7 scFv (SSS-S) H P238SCH2 WCH3 (10 ug/ml) forone hour on ice in PBS/2% FBS. Cells were washed in PBS/2% FBS andincubated with serial dilutions of 0.5 mg/ml FITC-CD16 for one hour onice. Cells were washed and specific binding measured by flow cytometryusing a Beckman-Coulter Epics C machine. Results were analyzed usingExpo analysis software and normalized fluorescence units graphed as afunction of concentration. FIG. 76 illustrates an experiment on theability of 2H7 scFv (SSS-S) H WCH2 WCH3 and 2H7 scFv (SSS-S) H P238SCH2WCH3 constructs to bind U937 cells. U937 cells (106) expressing CD64were incubated in PBS/2% FBS for one hour on ice with 2H7 scFv (SSS-S) HWCH2 WCH3 or 2H7 scFv (SSS-S) H P238SCH2 WCH3. Cells were washed andincubated for one hour on ice with FITC-goat anti-human IgG1 (Fcspecific) (Caltag) at a final dilution of 1:100. Cells were washed andfluorescence analyzed on a Beckman-Coulter EpicsC flow cytometer. Datawas analyzed using Expo analysis software, and fluorescence intensitygraphed as a function of the concentration of 2H7 scFv (SSS-S) H WCH2WCH3 or 2H7 scFv (SSS-S) H P238SCH2 WCH3. This figure shows that boththe 2H7 scFv (SSS-S) H WCH2 WCH3 construct and the 2H7 scFv (SSS-S) HP238SCH2 WCH3 construct bound to U937 cells with equivalent titrationcurves. The results indicated that the 2H7 scFv (SSS-S) H P238SCH2 WCH3construct was not impaired in its binding to the high affinity Fcreceptor FcγRI (CD64). The fact that binding to high affinity FcγRI onU937 cells was similar for the two molecules in this experimentindicated that the P238S amino acid change selectively reduced thebinding to FcγRIII. FIG. 77 illustrates B cell depletion in macaquesmediated by a anti-CD20 construct (2H7 scFv (SSS-S) H WCH2 WCH3) and aanti-CD20) scFv with a CH2 domain mutation. 2H7 scFv (SSS-S) H WCH2 WCH3or 2H7 scFv (SSS-S) H P238SCH2 WCH3 were administered to macaques (M.fasicularis) by intravenous injection at 6 mg/kg, with two infusionsgiven one week apart. The effect on circulating B cells was measured bydetection of CD40 positive B cells in peripheral blood. Blood sampleswere drawn from injected animals at days 7, 0, 1, 3, 7, 8, 10, 14, 28,and 43. B cell depletion was estimated by performing CBC (complete bloodcounts) and two-color flow cytometry analysis on monkey blood. FITC orPE conjugates of antibodies against CD40, CD19, CD20, IgG, CD3, CD8 wereused in various combinations. Data are plotted as the number of CD40positive blood B cells tabulated in thousands of cells per microliterover time relative to the initial pre-injection time point level of Bcells (maximum). This experiment showed that 2H7 scFv (SSS-S) H WCH2WCH3 scFv injection resulted in rapid and complete depletion ofcirculating B cells that lasted longer than 28 days following the secondinjection. Injection of 2H7 scFv (SSS-S) H P238SCH2 WCH3 scFv did notcompletely deplete B cells even though CD20 epitopes were saturated. Aslow reduction in B cells to approximately 50% of initial levels wasobserved during the first 2 weeks, but B cells rapidly returned tostarting levels in these animals. These results indicate that theability to bind high affinity FcγRI and to mediate complement dependentcytotoxicity is not sufficient for a CytoxB20G molecule to completelydeplete circulating B cells, and that ADCC mediated by interaction withCD16 is likely necessary for rapid and sustained B cell depletion.

These experiments indicated that the 2H7 scFv (SSS-S) H WCH2 WCH3 scFvsare able to trigger B cell depletion functions through three differentmechanisms of action (1) induction of apoptosis, (2) CDC effectormechanisms, and (3) ADCC effector mechanisms. All three of thesemechanisms probably contribute to depletion of B cells in vivo. The dataindicate that complete and sustained depletion of B cells requiresintact ADCC effector mechanisms. However, the partial depletion obtainedwith an ADCC negative mutant also indicates that apoptosis and CDCmechanisms contribute to mediating a portion of the total B celldepletion effects. The results support the use of 2H7 scFv (SSS-S) HWCH2 WCH3 scFv in patients with a CD20 positive B cell malignancy orautoimmune disease.

Example 162 Induction of Apoptosis in B Cells by Anti-CD37 GS-1 VL11SscFv (SSS) H WCH2WCH3 Constructs

In this Example, anti-CD37 GS-1 VL11S scFv (SSS) H WCH2WCH3 constructswere evaluated for their ability to bind target cells and for theirability to induce apoptosis. The constructs were also physicallycharacterized by size-exclusion chromatography (SEC). FIG. 78illustrates the SEC profile of G28-1 (anti-CD37) constructs having SSChinge domain forms (GS-1 VL11S scFv (SSC) H WCH2WCH3). G28-1 (anti-CD37)constructs were purified from CHO culture supernatants by Protein Aaffinity chromatography. Purified aliquots of 10-25 mg were subjected toHPLC over a Tosoh Biosep, Inc. TSK 3000 SWXL HPLC column, pore size 5mm. The flow rate was 1 ml/min, in PBS, pH 7.2 running buffer. Migrationrates of molecular weight standards are indicated below the tracing. TheGS-1 VL11S scFv (SSS) H WCH2WCH3 construct is indicated in blue, whilethe GS-1 VL11S scFv (SSC) H WCH2WCH3 construct is indicated in red. TheGS-1 VL11S scFv (SSS) H WCH2WCH3 construct generated a uniform peak ofapproximately 75-100 kDa, while the GS-1 VL11S scFv (SSC) H WCH2WCH3construct generated a smaller form and other heterogeneous forms,including a high molecular weight form greater than 200 kDa. FIG. 79illustrates the binding of G28-1 (anti-CD37) constructs to B celllymphoma cell lines. Serial dilutions of purified GS-1 VL11S scFv (SSS)H WCH2WCH3 or GS-1 VL11S scFv (SSC) H WCH2WCH3 were incubated with 10⁶cells of each cell type for 60 minutes on ice in PBS/2% FBS. Sampleswere washed twice, and incubated with a mixture of FITC goat anti-humanIgG and FITC goat anti-human IgG F(ab′)₂ (CalTag) at 1:100 each, on icefor 45 minutes. Samples were washed and analyzed by flow cytometry usinga FACsCalibur (Becton-Dickinson). The results of this experiment showthat both GS-1 VL11S scFv (SSS) H WCH2WCH3 and GS-1 VL11S scFv (SSC) HWCH2WCH3 constructs bound to BJAB and Ramos cells, and moderately toWIL-2 cells. G28-1 (anti-CD37) (SSS) H G scFv or G28-1 (SSC) H G scFvconstructs molecules bound Raji and Namalwa cells at lower levels inthese experiments. Binding to all lines was significant becausebackground fluorescence has been subtracted from the data shown in FIG.79. FIG. 80 illustrates the binding of annexin and v-propidium iodide toRamous cells after overnight incubation with GS-1 VL11S scFv (SSS) HWCH2WCH3 or GS-1 VL11S scFv (SSC) H WCH2WCH3 forms of scFvs. AnnexinV-PIstaining of Ramos Cells incubated for 24 hours with G28-1 (anti-CD37)scFvs. Ramos B cells at 10⁶ cells/ml were incubated in 12 well dishesfor 24 hours with G28-1 (anti-CD37) scFvs at 10 mg/ml, in a total volumeof 2 ml. Cells were stained with annexinV and propidium iodide with akit obtained from Immunotech, according to manufacturer's instructions.Samples were analyzed by two-color flow cytometry using a FACsCaliburflow cytometer (Becton-Dickinson). The % of the total cells in eachquadrant is indicated next to each dot plot. FIG. 83 shows the bindingof Annexin V-Propidium Iodide to cells after overnight incubation withGS-1 VL11S scFv (SSS) H WCH2WCH3 or GS-1 VL11S scFv (SSC) H WCH2WCH3.The results indicated that both GS-1 VL11S scFv (SSS) H WCH2WCH3 andGS-1 VL11S scFv (SSC) H WCH2WCH3 constructs were able to induceapoptosis, but that the SSC form induced more apoptosis than the SSSform. FIG. 81 illustrates the inhibition of proliferation of Ramos cellsgrown in the presence of G28-1 (anti-CD37) constructs. Ramos B cellswere incubated with serial dilutions of purified G28-1 (anti-CD37)constructs containing either the IgG1 hinge identified as (SSS) H or(SSC) H. Cultures were incubated in 96 well flat bottom tissue culturedishes (Costar) at 37° C., 5% CO₂ for 36 hours prior to pulsing with3H-thymidine for the last 12 hours of a 48 hour incubation (0.75mCi/well). Cells were harvested onto 96-well GFC plates using a Packardharvester, dried, and 25 ml Microscint scintillation fluid added to eachwell prior to counting on a TopCount NXT microplate (Packard)scintillation counter. Data are plotted as cpm incorporated versusprotein concentration. Each construct showed increasing inhibition ofproliferation with increasing protein concentration. FIG. 82 illustratesthe induction of apoptosis in Ramos B cells cultured in the presence ofand 2H7 (anti-CD20) and G28-1 (anti-CD37) constructs. Ramos B cells wereincubated with CD20 and/or CD37 targeted constructs (10 mg/ml) insolution for 20 hours. The cells were then harvested, washed, andincubated in annexinV and propidium iodide using a staining kit fromImmunotech prior to two color flow cytometry using a FACsCalibur flowcytometer (Becton-Dickinson). The graph shows the percentage of annexinV positive cells identified by their staining in the right quadrants ofthe dot plots. The results of this experiment indicate that the bothG28-1 constructs were more efficient than the 2H7 construct and that theG28-1 VL11S scFv (SSS) H WCH2 WCH3 construct was more efficient than theG28-1 VL11S scFv (SSC) H WCH2 WCH3 construct. However, the amount ofapoptosis of Ramos cells was greatest when the 2H7 and the G28-1constructs were used in combination. FIG. 83 illustrates the killing ofRamos cells by complement dependent cytotoxicity (CDC) induced by 2H7(anti-CD20) constructs and G28-1 (anti-CD37) constructs with hingeregion mutations. 2H7 scFv (CSS-S) H WCH2 WCH3 (anti-CD20), G28-1 scFv(SSS) H G, G28-1 (SCS)H (anti-CD37-scFv), G28-1 (CSS)H (anti-CD37-scFv),or G28-1 (SSC) H (anti-CD37-scFv) were incubated at 10 mg/ml with 10⁴Ramos Target Cells and a 1:10 dilution of rabbit complement (PelFreez)in a volume of 150 ml for 90 minutes. Aliquots were stained with trypanblue (Invitrogen), and counted using a hemacytometer to determine thepercentage of the cell population killed during treatment. Negativecontrols with cells and only one reagent were also included. Both G28-1(SSS) (anti-CD37 scFv) and G28-1 (SSC) (anti-CD37 scFv) rapidly killedRamos cells in the presence of rabbit complement in this experiment. Theactivity of G28-1 (SSC) (anti-CD37 scFv) was higher than that of G28-1(SSS) (anti-CD37 scFv), and was nearly as potent in this assay as theCD20-directed 2H7 scFv (CSS-S)H WCH2 WCH3 scFv molecule. FIG. 84illustrates the induction of ADCC of Ramos cells incubated with G28-1(anti-CD37) scFvs. G28-1 (anti-CD37) constructs at 10 mg/ml wereincubated in flat-bottom 96 well plates with 10⁴ ⁵¹Cr-labeled Ramoscells and resting human PBMCs at different effector:target ratiosranging from 0 to 100. All incubations were performed in triplicate ateach effector:target ratio. Natural killing was measured at eacheffector:target ratio by omission of the constructs. Spontaneous releasewas measured without addition of PBMC or fusion protein, and maximalrelease was measured by the addition of detergent (1% NP-40) to theappropriate wells. Reactions were incubated for 6 hours, and 100 mlculture supernatant harvested to a Lumaplate (Packard Instruments) andallowed to dry overnight prior to counting cpm released on a Packard TopCount NXT Microplate Scintillation Counter. Results from theseexperiments indicated that the G28-1 (anti-CD37) constructs bind totarget cells, that they induce apoptosis in the B cell lines, and thatthey mediate effector functions including CDC and ADCC.

Additional non-limiting representative constructs or sequences within,or useful within, the present inventions are as follows:

HuIgG1 wild type hinge. CH2, CH3 (nucleotide sequence) (SEQ ID NO: 1)tctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga HuIgG1 wild type hinge, CH2, CH3(amino acid sequence) (SEQ ID NO: 2)SDQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKLlama IgG1 hinge, CH2, CH3 (nucleotide sequence) (SEQ ID NO: 3)tgatcaagaaccacatggaggatgcacgtgcccncagtgcccncaatgcccttgcnccngaactccaggaggcccttctgtctttgtcttccccccgaaacccaaggacgtcctctccatttttggaggccgagtcacgtgcgttgtagtggacgtcggaaagaaagaccccgaggtcaatttcaactggtatattgatggcgttgaggtgcgaacggccaatacgaagccaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaggaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagaactggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccagctgacatcaacgttgagtggcagaggaacggtcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcggggagaaaccttaacctgtgtggtgatgcatgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctaga Llama IgG1 hinge, CH2, CH3(in FIG. 23 as Llama IgG1 (amino acid sequence) (SEQ ID NO: 4)EPHGGCTCPQCPAPELPGGPSVFFPPKPKDVLSISGRPEVTCVVVDVGKEDPEVNFNWYDGVEVRTANTKPKEEQFNSTYRVVSVLPIQHQDWLTGKEFKCKVNNKALPAPIERTISKAKGQTREPQVYTLAPHREELAKDTVSVTCLVKGFYPADINVEWQRNGQPESEGTYANTPPQLDNDGTYFLYSRLSVGKNTWQRGETLTGVVMHEALHNHY TQKSITQSSGK LlamaIgG2 (nucleotide sequence) (SEQ ID NO: 5)tgatcaagaacccaagacaccaaaaccacaaccacaaccacaaccacaacccaatcctacaacagaatccaagtgtcccaaatgtccagcccctgagctcctgggagggccctcagtcttcatcttccccccgaaacccaaggacgtcctctccatttctgggaggcccgaggtcacgtgcgttgtggtagacgtgggccaggaagaccccgaggtcagtttcaactggtacattgatggcgctgaggtgcgaacggccaacacgaggccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaagaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccacctgatatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacytacgccaccacgccaccccagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcagggagaaaccttcacctgtgtggtgatgcacgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctaga.Llama IgG2 (amino acid sequence) (SEQ ID NO: 6)DQEPKTPKPQPQPQPQPNPTTESKCPKCPAPELLGGPSVFIFPPKYKDVLSISGRPEVTCVVVDVGQEDPEVSFNWYIDGAEVRTANTRPKEEQFNSTYRVVSVLPIQHQDWLTGKEFKCKVNNKALPAPIEKTISKAKGQTREPQVYTLAPHREELAKDTVSVTCLVKGFYIPDINVEWQRNGQPESEGTYATTPPQLDNDGTYFLYSKLSVGKNTWQQGETFTCVVMHIEALHNHYTQKSITQSSGK Llama IgG3 Fc (nucleotide sequence) (SEQ IDNO: 7)tgatcaagcgcaccacagcgaagaccccagctccaagtgtcccaaatgcccaggccctgaactccttggagggcccacggtcttcatcttccccccgaaagccaaggacgtcctctccatcacccgaaaacctgaggtcacgtgcttgtggtggacgtgggtaaagaagaccctgagatcgagttcaagctggtccgtggatgacacagaggtacacacggctgagacaaagccaaaggaggaacagttcaacagcacgtaccgcggtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccagcccccatcgagaggaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacctgcctggcaaaggcttcttcccagctgacatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgggacctacttcctctacagcaaactctccgtgggaaagaacacgtggcagcagggagaagtcttcacctgtgtggtgatgcacgaggctctacacaatcactccacccagaaatccatcacccagtctttcgggtaaatagtaatctagagggccc Llama IgG3 Fc(amino acid sequence) (SEQ ID NO: 8)DQAHHSEDPSSKCPKCPGPELLGGPTVFIFPPKAKDVLSITRKPEVTCLWWTWVKKTLRSSSSWSVDDTEVHTAETKPKEEQFNSTYRVVSVLPIQHQDWLTGKEFKCKVNNKALPAPERTISKAKGQTREPQVYTLAPHREELAKDTVSVTCLVKGFFPADINVEWQRNGQPESEGTYANTPPQLDNDGTYFLYSKLSVGKNTWQQGEVFTCVVMHEA LHNHSTQKSITQSSGK.HuIgG1 wild type hinge (nucleotide sequence) (SEQ ID NO: 9)gatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagca HuIgG1 wild typehinge (amino acid sequence) (SEQ ID NO: 10) DQEPKSGDKTHTCPPCPA HuIgG1H2, wild type hinge with leu at second position (nucleotide sequence)(SEQ ID NO: 11) gatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcaHuIgG1 H2, wild type hinge with leu at second position (amino acidsequence) (SEQ ID NO: 12) DLEPKSCDKTHTCPPCPA NTHuIgG1 wild type CH2(nucleotide sequence) (SEQ ID NO: 13)cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaHuIgG1 wild type CH2 (amino acid sequence) (SEQ ID NO: 14)PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKYNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPLEKTISKAK HuIgG1 wild typeCH3 (nucleotide sequence) (SEQ ID NO: 15)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaHuIgG1 wild type CH3 (amino acid sequence) (SEQ ID NO: 16)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDLAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HuIgG1 mutated hinge(C-C-C→S-S-S) (nucleotide sequence) (SEQ ID NO: 17)gatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagca HuIgG1 mutatedhinge (C-C-C→S-S-S) (amino acid sequence) (SEQ ID NO: 18)DQEPKSSDKTHTSPPSPA HIgG1MTH WTCH2CH3 (mutant hinge with wild type CH2and CH3 (reads from the hinge + Ig tail) (nucleotide sequence) (SEQ IDNO: 19)tgatcaccccaaatcttctgacaaaactcacacatctccaccgtcctcagcacctgaactcctgggtggaccgtcagtgttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcuctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataatctaga Mutant hinge, but wild type CH2 andCH3 (amino acid sequence) (SEQ ID NO: 20)DHPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 2H7scFv Ilama IgG1 (nucleotide sequence) (SEQ ID NO: 21)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagtcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaagaaccacatggaggatgcacgtgcccncagtgcccncagtgcccngcnccngaactnccaggaggcccttctgtctttgtcttccccccgaaacccaaggacgtcctctccatttttggaggccgagtcacgtgcgttgtagtggacgtcggaaagaaagaccccgaggtcaatttcaactggtatattgatggcgttgagtgcgaacggccaatacgaagccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaggaccatctccaaggccaaagggcagaccccgggagccgcaggtgtacaccctggccccacaccgggaagaactggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccagctgacatcaacgttgagtggcagaggaacggtcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcggggagaaaccttaacctgtgtggtgatgcatgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctaga2H7 scFv llama IgG1 (amino acid sequence) (SEQ ID NO: 22)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPHGGCTCQPCPAPELPGGPSVFVFPPKPKDVLSIFGGRVTCVVVDVGKKDPEVNFNWYIDGVEVRTANTKPKEEQFNSTYRVVSVLPIQHQDWLTGKEFKCKVNNKALPAPIERTISKAKGQTREPQVYTLAPHREELAKDTVSVTCLVKGFYPADINVEWQRNGQPESEGTYANTPPQLDNDGTYFLYSKLSVGKNTWQRGETLTCVVMHEALHNHYTQKSITQSS GK 2H7 scFv llamaIgG2 (nucleotide sequence) (SEQ ID NO: 23)aagcttgccgccsrggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttgttctctccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggacaacggtcaccgtctcttctgatcaagaacccaagacaccaaaaccacaaccacaaccacaaccacaacccaatcctacaacagaatccaagtgtcccaaatgtccagcccctgagctcctgggagggccctcagtcttcatcttccccccgaaacccaaggacgtcctctccatttctgggaggcccgaggtcacgtgcgttgtggtagacgtgggccaggaagacccgaggtcagtttcaactggtacattgatggcgctgaggtgcgaacggccaacacgaggccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaagaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggcccacaccgggaagagctggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccacctgatatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacytacgccaccacgccaccccagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcagggagaaacctcacctgtgtggtgatgcacgaggccctgcacaaccactacaccagaaatccatcacccagtcttcgggtaaatagtaatctaga AA2H7 scFv llama IgG2 (amino acid sequence)(SEQ ID NO: 24)MDFQVQIFSFLLISASVILARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSSPKPWIYAPSNLASGVPARFSGSGSGTSYLTISRVEAEDAATYYCQQWSFNPPTFGAGTKKLELKDGGGSGGGGSGGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWTGTGTTVTVSSDQEPKTPKPQPQPQPQPNPTTESKCPKCPAPEKKGGPSVFIFPPKPKDVLSISGRPEVTCVVVDVGQEDPEVSFNWYIDGAEVRTANTRPKEEQFNSTYRVVSVLPIQHQDWLTGKEFKCKVNNKALPAPIEKTISKAKGQTREPQVYTLAPHREELAKDTVSVTCLVKGFYPPDINVEWQRNGQPESEGTYATTPPQLDNDGTYFLYSKLSVGKNTWQQGETFTCVVMHE ALHNHYTQKSITQSSGKNT2H7 scFv llama IgG3 (nucleotide sequence) (SEQ ID NO: 25)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaagcgaaccacagcgaagaccccagctccaagtgtcccaaatgcccaggccctgaactccttggagggcccacggtcttccccccgaaagccaaggacgtcctctccatcacccgaaaacctgaggtcacgtgcttgtggtggacgtgggtaaagaagaccctgagatcgagttcaagctggtccgtggatgacacagaggtacacagaggtacacacggctgagacaa2H7 scFv llama IgG3 (amino acid sequence) (SEQ ID NO: 26)MDFQVQIFSFLLISASVILARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQAHSHSEDPSSKCPKCPGPELLGGPTVFIFPPKAKDVLSITRKPEVTCLWWTWVKKTLRSSSSWSVDDTEVHTAETKPKEEQFNSTYRVVSVLPIQHQDWLTQKEFKCKVNNKALPAPIERTISKAKGQTREPQVYTLAPHREELAKDTVSVTCLVKGFFPADINVEWQRNGQPESEGTYANTPPQLDNDGTYFLYSKLSVGKNTWQQGEVETCVVMHEALHNHSTQK SITQSSGK 2H7 scFvWTH WTCH2CH3 (nucleotide sequence) (SEQ ID NO: 27)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagtcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaoctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7 scFvWTH WTCH2CH3 (amino acid sequence) (SEQ ID NO: 28)MDFQVQIFSFLLISASVILARGQIVLSQSPAILSASPGEKVTMMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYYFDVWGTGTTVTVSSDQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFKYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK NTCD80transmembrane domain and cytoplasmic tail (+ restriction sites)(nucleotide sequence) (SEQ ID NO: 29)gcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatCD80 transmembrane domain and cytoplasmic tail (amino acid sequence)(SEQ ID NO: 30) ADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV40.2.220 VL (anti-human CD40 scFv #1-VL) (nucleotide sequence) (SEQ IDNO: 31)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataagtccagaggagtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagctagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacgg 40.2.220 VL (anti-human CD40scFv #1--VL) (amino acid sequence) (SEQ ID NO: 32)MDFQVQWSFLLISASVIMSRGVDWLTQSPATLSVTPGDRVSLSCPASQSISDYLHWYQQKSHESPRLLIKYASHSISGIPSRFSGSGSGSDFTLSTNSVEPEDVGIYYCQHGHSFPWTFGGGTKLEIKR 40.2.220 VH (for anti-human CD40 scFv #1-VH) (nucleotidesequence) (SEQ ID NO: 33)cagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagctccgggaatggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatca 40.2.220 VH (for anti-human CD40 scFv #1--VH)(amino acid sequence) (SEQ ID NO: 34)QIQLVQSGPELKKPGETVRISCKASGYAFTTTGMQWVQEMPGKGLKWIGWINTPLWSAKICRRLQGRFAFSLETSANTAYLQISNLKDEDTATYFCVRSGNGNYDLAYFA YWGQGTLVTVS40.2.220 scFv (anti-human CD40 scFv #1) (nucleotide sequence) (SEQ IDNO: 35)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttcgatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatca 40.2.220 scFv (anti-human CD40 scFv #1) (aminoacid sequence) (SEQ ID NO: 36)MDFQVQWSFLLISASVIMSRGVDIVLTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASHSISGIPSRFSGSGSGSDFTLSIINSVEPEDVGIIYYCQHGHSFPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQIQLVQSGPELKKPGETVRISCKASGYAFTTTGMQWVQEMPGKGLKWIGWINTPLWSAKICRRLQGRFAFSLETSANTAYLQISNLKDEDTATYFCVRSGNGNYDLAYFAYWGQGTLVTVS 2e12 VL (with L6 VK leaderpeptide) (nucleotide sequence) (SEQ ID NO: 37)atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacgg 2e12 VL (with L6 VKleader peptide) (amino acid sequence) (SEQ ID NO: 38)MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATLSCRASESVEYYVTSLMQWYQQKIPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKR 2e12 VH (no leader peptide) (nucleotide sequence)(SEQ ID NO: 39)caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaacucauactatgttatggactactgggtcaaggaacctcagtcaccgtctcctca(gatctg) 2e12 VH (amino acid sequence) (SEQ ID NO: 40)QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKNSLQTDDTARYYCARDGYSNFHYYVMD YWGQGTSVTVSS2e12scFv(+Restriction sites) (nucleotide sequence) (SEQ ID NO: 41)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattccagaggagtcgacattgtctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggcgtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctct(gatcag) 2e12scFv (amino acidsequence) (SEQ ID NO: 42)MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYCARDGYSNFHYYVMDYWGQGTSVTVSS 10A8 is anti-CD152(CTLA-4) 10A8 VL (with L6 VK leader peptide) (nucleotide sequence) (SEQID NO: 43)atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaacttattattgtcaacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacgg 10A8 VL (amino acid sequence)(SEQ ID NO: 44)MDFQVQIFSFLLISASVIMSRGVDIQMTQSPSSLSASLGGKVTITCKASQDIKKYIGWYQHKPGKGPRLLIYYTSTLQPGIPSRFSGSGSGRDYSLSIRNLEPEDIATYYCQQYDNLPLTFGSGTKLEIKR 10A8 VH (no leader peptide) (nucleotide sequence) (SEQID NO: 45)gatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatggatgggccacataagccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaaggaacctcagtcaccgtctcctctgatca 10A8 VH (amino acid sequence) (SEQ ID NO: 46)DVQLQEFGPGLVKPSQSLSLTCSVTGYSITSGFYWNWIRQFPGNKLEWMGHISHDGRNNYNPSLINRISITRDTSKNQFFLKLSSVTTEDTATYFCARHYGSSGAMDYWGQ GTSVTVSS 10A8SCFV (nucleotide sequence) (SEQ ID NO: 47)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaacttattattgtcaacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggtttctactggaactggatccgccagtttccgggaaacaaactggaatggatgggccacataagccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaggaacctcagtcctactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaggaacctcagtcaccgtctcctctgatca 10A8 SCFV (amino acid sequence) (SEQ ID NO: 48)MDFQVQIFSFLLISASVIMSRGVDIQMTQSPSSLSASLGGKVTITCKASQDIKKYIGWYQHKPGKGPRLLIYYTSTLQPGIPSRFSGSGSGRDYSLSIRNLEPEDIATYYCQQYDNLPLTFGSGTKLEIKRGGGGSGGGGSGGGGSDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGFYWNWIRQFPGNKLEWMGHISHDGRNNYNPSLINRISITRDTSKNQFFLKLSSVTTEDTATYFCARHYGSSGAMDYWGQGTSVTVSSD 40.2.220-hmtIgG1-hCD80(nucleotide sequence) (SEQ ID NO: 49)aagtcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccacctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat 40.2.220-hmtIgG1-hCD80 (amino acidsequence) (SEQ ID NO: 50)MDFQVQIFSFLLISASVIMSRGVDIVLTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASHSISGIPSRFSGSGSGSDFTLSINSVEPEDVGIYYCQHGHSFPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQIQLVQSGPELKKPGETVRISCKASGYAFTTTGMQWVQEMPGKGLKWIGWINTPLWSAKICRRLQGRFAFSLETSANTAYLQISNLKDEDTATYFCVRSGNGNYDLAYFAYWGQGTLVTVSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALOAOIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 2e12scFv-hmtIgG1-CD80fusion protein (nucleotide sequence) (SEQ ID NO: 51)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgcccgcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat2e12scIFv-hmtIgG1-CD80 fusion protein (amino acid sequence) (SEQ ID NO:52) MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQOOKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWWSNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYVFAPRCRERRRNERLRRESVRPV 10A8scFv-hmtIgG1-CD80 (nucleotide sequence) (SEQ ID NO: 53)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaacttattattgtcaacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacggggtggcggtggctcgggcggtggtgggtcgggtggcgggcggatctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatggatgggccacataagccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaggaacctcagtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctaccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat 10A8 scFv-hmtIgG1-CD80 (amino acid sequence)(SEQ ID NO: 54)MDFQVQIFSFLLISASVIMSRGVDIQMTQSPSSLSASLGGKVTITCKASQDIKKYIGWYQHKPGKGPRLLIYYTSTLQPGIPSRFSGSGSGRDYSLSIRNLEPEDIATYYCQQYDNLPLTFGSGTKLEIKRGGGGSGGGGSGGGGSDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGFYWNWIRQFPGNKLEWMGHISHDGRNNYNPSLINRISITRDTSKNQFFLKLSSVTTEDTATYFCARHYGSSGAMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 500A2-hmtIgG1-CD80(nucleotide sequence) (SEQ ID NO: 55)atgttgtatacatctcagctccttgggcttttactcttctggatttcagcctccagaagtgacatagtgctgactcagactccagccactctgtctctaattcctggagaaagagtcacaatgacctgtaagaccagtcagaatattggcacaatcttacactggtatcaccaaaaaccaaaggaggctccaagggctctcatcaagtatgcttcgcagtccattcctgggatcccctccagattcagtggcagtggttcggaaacagatttcactctcagcatcaataacctggagcctgatgatatcggaatttattactgtcaacaaagtagaagctggcctgtcacgttcggtcctggcaccaagctggagataaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtcaagctgcagcagtccggttctgaactagggaaacctggggcctcagtgaaactgtcctgcaagacttcaggctacatattcacagatcactatatttcttgggtgaaacagaagcctggagaaagcctgcagtggataggaaatgtttatggtggaaatggtggtacaagctacaatcaaaaattccagggcaaggccacactgactgtagataaaatctctagcacagcctacatggaactcagcagcctgacatctgaggattctgccatctattactgtgcaagaaggccggtagcgacgggccatgctatggactactggggtcaggggatccaagttaccgtctcctctgatctggagcccaaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagtccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat 500A2-hmtIgG1-CD80 (amino acid sequence) (SEQ ID NO:56) MLYTSQLLGLLLFWISASRSDIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPSRFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIKRGGGGSGGGGSGGGGSQVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSYNQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 2H7 scFv MTH(SSS)WTCH2CH3(nucleotide sequence) (SEQ ID NO: 57)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgtcgctcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagacctctccctgtctccgggtaaatgatctaga 2H7 scFvMTH(SSS)WTCH2CH3 (amino acid sequence) (SEQ ID NO: 58)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSCSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSTSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYKQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 2H7 scFvIgAH IGG WT CH2CH3 (2H7 scFv with IgA hinge and WT CH2 and CH3)(nucleotide sequence) (SEQ ID NO: 59)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgcgcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcacctgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7 scFv 1gAH IGG WT CH2CH3 (amino acid sequence) (SEQ ID NO: 60)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSDQPVPSTPPTPSPSTPPTPSPSCAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIELTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 2H7 scFvIgAH IgACH2CH3 (2H7 scFv IgAhinge and IgA CH2 and CH3) (nucleotidesequence) (SEQ ID NO: 61)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctccccaaaccctggatttatgccccatccaacctggcttctggagtcccatctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgagctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgatggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaaggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacggcacctgctactgataatctaga 2H7 scFv IgAH IgACH2CH3 (2H7 scFv IgAhinge and IgA CH2 and CH3) (amino acid sequence) (SEQ ID NO: 62)MDFQVQIFSFLLISASVIIARGQWLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKIPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNBLVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSRRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDCTCY IgA hinge-CH2-CH3 (Human IgA tail, full length)(nucleotide sequence) (SEQ ID NO: 63)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacggcacctgctactgataatctaga IgA hinge-CH2-CH3 (Human IgA tail, full length) (amino acidsequence) (SEQ ID NO: 64)DQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHZRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKISAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNBLVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY Human J Chain (nucleotide sequence) (SEQID NO: 65)agatctcaagaagatgaaaggatgttcttgttgacaacaaatgtaagtgtgcccggattacttccaggatcatccgttcttccgaagatcctaatgaggacattgtggagagaaacatccgaattattgttcctctgaacaacagggagaatatctctgatcccacctcaccattgagaaccagatttgtgtaccatttgtctgacctcagctgtaaaaaatgtgatcctacagaagtggagctggataatcagatagttactgctacccagagcaatatctgtgatgaagacagtgctacagagacctgctacacttatgacagaaacaagtgctacacagctgtggtcccactcgtatatggtggtgagaccaaaatggtggaaacagccttaaccccagatgcctgctatcctgactaatctagaHuman J Chain (amino acid sequence) (SEQ ID NO: 66)RSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIVPLNNRENISDPTSPLRTRFVYHLSDLSCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYP 4 carboxy terminal amino acids deleted from IgACH3 (amino acid sequence) (SEQ ID NO: 67) GTCY IgAH IgAT4 (human IgAtail, truncated (3T1)-(missing last 4 amino acids from carboxy terminus)(nucleotide sequence) (SEQ ID NO: 68)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccaccccgactgtcactgcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcagtgtcacctcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctagaIgAH IgAT4 (amino acid sequence) (SEQ ID NO: 69)DQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVD 2H7 scFv IgAH IgAT4 (2H7 scFv IgA 3T1construct; truncates the CH3 domain at the 3′end) (nucleotide sequence)(SEQ ID NO: 70)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggcaagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagtttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaaggggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtgctgttgtcatggcggaggtggactgataatctaga 2H7 scFv IgAH-T4 (amino acid sequence) (SEQ ID NO:71) MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLA GKPTHVNVSVVMAEVD14 amino acids deleted from IgAH-T4 (so that total of 18 amino acidsdeleted from wild type IgA CH3) (amino acid sequence) (SEQ ID NO: 72)PTHVNVSVVMAEVD IgAH IgA-T18 (human IgA Tail truncated, 3T2) (nucleotidesequence) (SEQ ID NO: 73)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcccaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaa IgAH IgA-T18 (amino acid sequence) (SEQID NO: 74) DQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAF TQKTIDRLAGK 2H7scFv IgAH IgAT18 (human IgA Tail truncated, 3T2) (nucleotide sequence)(SEQ ID NO: 75)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcaactccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaa 2H7 scFv IgAHIgAT18 (amino acid sequence) (SEQ ID NO: 76)MDFQVQIFSFLLISASVILARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLA GK CTLA-4 IgGWTH WTCH2CH3 (human-oncoMLP-CTLA4EC-hIgGWT) (nucleotide sequence) (SEQID NO: 77)gcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcaacccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaCTLA-4 IgG WTH WTCH2CH3 (amino acid sequence) (SEQ ID NO: 78)MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTVRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human OncoM leaderPeptide + CTLA4 EC (BclI) (nucleotide sequence) (SEQ ID NO: 79)atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgcagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgaacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcaa Human OncoM leader Peptide + CTLA4 EC (aminoacid sequence) (SEQ ID NO: 80)MGVLLTTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGLASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQ Human OncoM leader (nucleotidesequence) (SEQ ID NO: 81)atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatgHuman OncoM leader (amino acid sequence) (SEQ ID NO: 82)MGVLLTQRTLLSLVLALLFPSM Human CTLA4 EC (no LP) (nucleotide sequence) (SEQID NO: 83)gcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtcgtgcggcaacctacatgacggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattct Human CTLA4 EC (no LP) (amino acidsequence) (SEQ ID NO: 84)AMHVAQPAVVLASSRGLASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMTGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNG TQIYVIDPEPCPDSHuman CTLA4 IgG MTH (SSS) MTCH2CH3 (nucleotide sequence) (SEQ ID NO: 85)atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctgcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcaacccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga Human CTLA4 IgG MTH (SSS)MTCH2CH3 (amino acid sequence) (SEQ ID NO: 86)MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGLASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CTLA-4 IgAH IgACH2CH3(human-oncoMLP-CTLA4EC-IgA) (nucleotide sequence) (SEQ ID NO: 87)atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagactgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacggcacctgctactgataatctaga CTLA-4 IgAH IgACH2CH3 (amino acid sequence)(SEQ ID NO: 88) MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCABPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLATGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVS VVMACVDGTCYCTLA-4 IgAH IgA-T4 (human-oncoMLP-CTLA4EC-IgA3T1) (nucleotide sequence)(SEQ ID NO: 89)atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgaaaagtacctgacttgggcatcccggcaggagcccagcaagggcaccaccaccttcgctgtgaccagcatactgcgcgaggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctaga CTLA-4 IgAH IgA-T4 (amino acid sequence) (SEQ ID NO: 90)MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLATGFSPKDVLVRWLQGSQELPRELYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVS VVMAEVD humanIgG1 CH2 with 238 mutation pro→ser (nucleotide sequence) (SEQ ID NO: 91)cctcgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaghuman 1gG1 CH2 with 238 mutation pro→ser (amino acid sequence) (SEQ IDNO: 92) PELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK Amino acidssurrounding Pro to Ser in CH2 (amino acid sequence) (SEQ ID NO: 93)PAPELLGGPS Amino acids surrounding Pro to Ser in CH2 (amino acidsequence) (SEQ ID NO: 94) PAPELLGGSS hIgE3BB (leaves an open readingframe at end of gene to read into transmembrane and cytoplasmic taildomain attached at either the BamHI or SfuI sites) (nucleotide sequence)(SEQ ID NO: 95) gtt gtt ttc gaa gga tcc gct tta ccg gga ttt aca gac accgct cgc tgg human IgE Fc (CH2-CH3-CH4) ORF (nucleotide sequence) (SEQ IDNO: 96)tgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctgcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaahuman IgE Fc (CH2-CH3-CH4) ORF (amino acid sequence) (SEQ ID NO: 97)DHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTLLCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKADPS IFhIgGwtBc15(nucleotide sequence) (SEQ ID NO: 98) gtt gtt tga tca gga gcc caa atcttg tga caa aac tca cac atg ccc acc gtg ccc agc acc (63 mer) HuIgGMHWC(sense, 5′ primer for mutating wild type hinge CCC to mutant SSS)(nucleotide sequence) (SEQ ID NO: 99) gtt gtt gat cag gag ccc aaa tcttct gac aaa act cac aca tct cca ccg tcc cca gca cct gaa ctc ctg ggt ggaccg tca gtc ttc c 1D8 VH (nucleotide sequence) (SEQ ID NO: 100)caggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctct1D8 VH (no leader) (amino acid sequence) (SEQ ID NO: 101)QVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVT VSS 1D8 VL (noleader) (nucleotide sequence) (SEQ ID NO: 102)gacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacgg 1D8 VL (aminoacid sequence) (SEQ ID NO: 103)DIVLTQSPTTIAASPGEKVTTTCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKR 1D8 scFv (nucleotidesequence) (SEQ ID NO: 104)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatc1D8 scFv (amino acid sequence) (SEQ ID NO: 105)MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTOLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSS 1D8 scFv IgG WTH WTCH2CH3 (nucleotidesequence) (SEQ ID NO: 106)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactgggggccaaggagtcatggtcacagtctcctctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacacccccatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 1D8 scFv IgG WTH WTCH2CH3 (amino acidsequence) (SEQ ID NO: 107)MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWTYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNYTQKSLSLSPGK 1D8 scFv IgG MTHMTCH2CH3-CD80 (nucleotide sequence) (SEQ ID NO: 108)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacaagcccaccgagcccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata 1D8 scFv IgG MTH MTCH2CH3-CD80 (amino acid sequence) (SEQ IDNO: 109) MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWTYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 1D8 scFv IgG WTH WTCH2CH3-CD80(nucleotide sequence) (SEQ ID NO: 110)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata 1D8 scFv IgG WTH WTCH2CH3-CD80 (amino acid sequence) (SEQ IDNO: 111) MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMTWYQQKSGASPKLWIYDTSKLASGVPNRFSDSDSDTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV Anti-human CD3 scFv WTHWTCH2CH3 (nucleotide sequence) (SEQ ID NO: 112)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgggagctcggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctatcgatgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgtcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAnti-human CD3 scFv WTH WTCH2CH3 (amino acid sequence) (SEQ ID NO: 113)MDFQVQIFSFLLISASVIMSRGVDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYTSRLHSGVPSRFSGSGSGTDYSLTAINLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRELGGGGGSGGGGSGGGGGSIDEVQLQQSGPELVKPGASMSCKASGYSFTGYIVNWLKQSHGKNLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSDQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYQ KSLSLSPGK2H7-antiCD40 scFv MTH (SSS) MTCH2WTCH3 (2h7-40.2.220Ig + restrictionsites) (nucleotide sequence) (SEQ ID NO: 114)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaatccaactctgaagaagcaaagaaagaggaggccaaaaaggaggaagccaagaaatctaacagcgtcgacattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcctactttgcttactgggggccaagggacactggtcactgtctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga2H7-antiCD40 scFv MTH (SSS) MTCH2WTCH3 (2H7-40.2.220Ig) (amino acidsequence) (SEQ ID NO: 115)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKGGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQSNSEEAKKEEAKKEEAKKSNSVDIVLTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASHSISGIPSRFSGSGSGSDFTLSINSVEPEDVGIYYCQHGHSFPAWTFGGGTKLEIKRGGGGSGGGGSGGGGSQIQLWQSGPELKKPGETVRISCKASGYAFTTTGMQWVQEMPGKGLKWIGWINTPLWSAKICRRLQGRFAFSLETSANTAYLQISNLKDEDTATYFCVRSGNGNYDLAYFAYWGQGTLVTVSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5B9 VH (includes the VHleader peptide) (nucleotide sequence) (SEQ ID NO: 116)atggctgtcttggggctgctcttctgcctggtgacatttccaagctgtgtcctatcccaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca5B9 VH (minus the leader) (nucleotide sequence) (SEQ ID NO: 117)caggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgotgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca 5B9 VH (includes leader peptide) (amino acid sequence)(SEQ ID NO: 118)MAVLGLLFCLVTFPSCVLSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCAIRNGGDNYPYYYAMDYWGQGTSVTVSS 5B9 VH (no leader peptide) (amino acidsequence) (SEQ ID NO: 119)QVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDY WGQGTSVTVSS 5B9VL (nucleotide sequence) (SEQ ID NO: 120)atgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacgg 5B9 VL (amino acid sequence)(SEQ ID NO: 121)MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKR 5B9 scFv (nucleotide sequence) (SEQ ID NO: 122)aagcttgccgccatgaggttctctgcccagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcacttttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctct 5B9 scFv (amino acid sequence)(SEQ ID NO: 123)MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSSGSGTDFTLRISRVEAVEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSS 5B9 scFv-hmtIgG1-hCD80(nucleotide sequence) (SEQ ID NO: 124)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcagggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacaagcccaccgagcccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgacgaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcagggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataatcgatactcgag 5B9scFv-hmtIgG1-hCD80 (amino acid sequence) (SEQ ID NO: 125)MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKGELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVFQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKSTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRRSQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 2e12 scFv WTHCH2 CH3 (2e12 scFv-WthIgG-CD80) (nucleotide sequence) (SEQ ID NO: 126)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgacgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaagtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat 2e12 scFv WTH CH2 CH3(2e12 scFv-WthIgG-CD80) (amino acid sequence) (SEQ ID NO: 127)MDQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLLSAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIEMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVIWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDCVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKSFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSQAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 2H7-human IgEFe (CH2-CH3-CH4) (nucleotide sequence) (SEQ ID NO: 128)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacaactcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctaga 2H7 scFv IgE (CH2-CH3-CH4) (amino acid sequence) (SEQ ID NO:129) MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKGGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSDHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYSLRPSPFDLFIRLSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK 2H7 scFv MH (SSS) MCH2WTCH3(nucleotide sequence) (SEQ ID NO: 130)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacagcgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7 scFvMH (SSS) MCH2WTCH3 (amino acid sequence) (SEQ ID NO: 131)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTIVTVSSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 5B9 scFvMTHWTCH2CH3 (nucleotide sequence) (SEQ ID NO: 132)aagcttgccgccatgagggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggaggtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttcttactgtgccagaaatgggggtgataactacccttattactatgctatggactacctggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga5B9 scFv MTHWTCH2CH3 (amino acid sequence) (SEQ ID NO: 133)MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK Human IgG1hinge mutations 2H7 scFv-MTH (CSS) WTCH2CH3 (nucleotide sequence) (SEQID NO: 134)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggaatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatggcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7scFv-MTH (CSS) WTCH2CH3 (amino acid sequence) (SEQ ID NO: 135)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSCDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 2H7 scFv-MTH(SCS) WTCH2CH3 (nucleotide sequence) (SEQ ID NO: 136)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaacctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatgcccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctgcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctcctcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7scFv-MTH (SCS) WTCH2CH3 (amino acid sequence) (SEQ ID NO: 137)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASGEKVTMTCRASSSVSYMHWYQQKKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 2H7scFv-MTH (SSC) WTCH2CH3 (nucleotide sequence) (SEQ ID NO: 138)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttcccaccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7scFv-MTH (SSC) WTCH2CH3 (amino acid sequence) (SEQ ID NO: 139)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTFRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK HIgGMHcys1(nucleotide sequence) (SEQ ID NO: 140) gtt gtt gat cag gag ccc aaa tcttct gac aaa act cac aca tg HIgGMHcys2 (nucleotide sequence) (SEQ ID NO:141) gtt gtt gat cag gag ccc aaa tct tgt gac aaa act cac aca tct cca ccgtgc HIgGMHcys3 (nucleotide sequence) (SEQ ID NO: 142) gtt gtt gat caggag ccc aaa tct tgt gac aaa act cac aca tgt cca ccg tcc cca gca cctHuIgG1 MTCH3Y405 (nucleotide sequence) (SEQ ID NO: 143)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaHuIgG1 MTCH3Y405 (amino acid sequence) (SEQ ID NO: 144)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFYLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HuIgG1 MTCH3A405(nucleotide sequence) (SEQ ID NO: 145)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaHuIgG1 MTCH3A405 (amino acid sequence) (SEQ ID NO: 146)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGFALYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HuIgG1 MTCH3A407(nucleotide sequence) (SEQ ID NO: 147)GggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaHuIgG1 MTCH3A407 (amino acid sequence) (SEQ ID NO: 148)GQPREPQVYTLPPSRESMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HuIgG1 MTCH3Y405A407(nucleotide sequence) (SEQ ID NO: 149)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaHuIgG1 MTCH3Y405A407 (amino acid sequence) (SEQ ID NO: 150)GQPREPQVYTLPPSREEMIKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFYLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HuIgG1 MTCH3A405A407(nucleotide sequence) (SEQ ID NO: 151)gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaHuIgG1 MTCH3A405A407 (amino acid sequence) (SEQ ID NO: 152)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALASKLTVDRSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2H7 scFv MTH (SSS)WTCH2MTCH3Y405 (nucleotide sequence) (SEQ ID NO: 153)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgatctaga 2H7 scFvMTH (SSS) WTCH2MTCH3Y405 (amino acid sequence) (SEQ ID NO: 154)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFYLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 2H7 scFv MTH(SSS) WTCH2MTCH3A405 (nucleotide sequence) (SEQ ID NO: 155)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga 2H7 scFv MTH(SSS) WTCH2MTCH3A405 (nucleotide sequence) (SEQ ID NO: 156)mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaelvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfalyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 2H7 scFv MTH (SSS) WTCH2MTCH3A407 (nucleotide sequence) (SEQ IDNO: 157)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga 2H7 scFv MTH(SSS) WTCH2MTCH3A407 (amino acid sequence) (SEQ ID NO: 158)MDFQVQWSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMIIWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSGKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYILPPSREEMTKNQVVSLTCLVKGYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLLS PGK 2H7 scFv MTH(SSS) WTCH2MTCH3Y405A407 (nucleotide sequence) (SEQ ID NO: 159)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttcagatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttcccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctacctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga 2H7 scFv MTH(SSS) WTCH2MTCH3Y405A407 (amino acid sequence) (SEQ ID NO: 160)MDFQVQIFSFLLISASVILAPGQIVLSQSPAILSASGEKVTMTCRESSSVSYMHHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLEIKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTDYNQKFKGKATLTVDVKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFYLASKLTVDKSRWQQGNVFSCSCMHEALHNHTQKSLSLS PGK 2H7 scFv MTH(SSS) WTCH2MTCH3A405A407 (nucleotide sequence) (SEQ ID NO: 161)aagcttgccgccatggattttcaagtgcagatatcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcccgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatogagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcggccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcgccctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga 2H7 scFv MTH(SSS) WTCH2MTCH3A405A407 (amino acid sequence) (SEQ ID NO: 162)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASGEKVTMCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 2H7 scFv MTH(SCC) WTCH2CH3 (nucleotide sequence) (SEQ ID NO: 163)aagcttgccgccatggattttcaagtgcagcttttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcccgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcaaaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcctcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtgacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7 scFvMTH (SCC) WTCH2CH3 (amino acid sequence) (SEQ ID NO: 164)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTCPPCPAPELLGGSPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 2H7 scFvMTH (CSC) WTCH2CH3 (nucleotide sequence) (SEQ ID NO: 165)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaagttgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatctccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaaggacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7 scFvMTH (CSC) WTCH2CH3 (amino acid sequence) (SEQ ID NO: 166)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYPCARVVYYSNSYWYFDVWGTGTTVTSSDQEPKSCDKTHTSPPCPAPELLGGPSVFCFFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSEFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 2H7 scFvMTH (CCS) WTCH2CH3 (nucleotide sequence) (SEQ ID NO: 167)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatgtccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggggtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga 2H7 scFvMTH (CCS) WTCH2CH3 (amino acid sequence) (SEQ ID NO: 168)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEOKSCDKTHTCPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVVSSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKHuIgAHIgA-T4-ORF (nucleotide sequence) (SEQ ID NO: 169)tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaac HuIgAHIgA-T4-ORF (amino acid sequence) (SEQ ID NO: 170)DQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDADPSN 1D8-IgAH IgA-T4-CD80 (nucleotidesequence) (SEQ ID NO: 171)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagcttataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatcagccagttccctcaactccacctaccccaactccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac AA 1D8 scFvIgAH IgA-T4-CD80 (amino acid sequence) (SEQ ID NO: 172)MDFQVQIFSFLLISASVLMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSIHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQPPDRDLCGCYCVSSVLPGCAEPWNHGKTFTCTAAYPEASKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARCFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDADPSNNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV human IgE Fc(CH2-CH3-CH4) ORF (nucleotide sequence) (SEQ ID NO: 173)tgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccaogcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaaAA human IgE Fc (CH2-CH3-CH4) ORF (amino acid sequence) (SEQ ID NO: 174)DHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGLASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGCSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKADPS 1D8 scFv-human IgEFc (CH2-CH3-CH4)-CD80 (nucleotide sequence) (SEQ ID NO: 175)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacaacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccaggaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata 1D8-scFv-human IgE Fc(CH2-CH3-CH4)-CD80 (amino acid sequence) (SEQ ID NO: 176)MDFQVQIFSFLLISASVLMSRGVDIVLTQSPTTJAASPGEKVTITCPASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPBGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKAKPSKLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 5B9-IgAH IgA-T4-CD80 (nucleotide sequence) (SEQ IDNO: 177)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgcttccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacagagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac 5B9-IgAH IgA-T4-CD80 (amino acid sequence) (SEQ ID NO:178) MRFSAQLLGLLVLWIRGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITSYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPPLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTIFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDADPSNNLLPSWAITLISVNGIFVVICCCTYCFAPRCRER RRNERCRRESVRPV5B9-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (nucleotide sequence) (SEQ IDNO: 179)aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccgcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatggaattttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata5B9-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (amino acid sequence) (SEQ IDNO: 180) MRFSAQLLGLLVLWLPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISKVEAEDVGVYYCAQNLELPLTFGAGTKTELKRGGGGSGGGGSGGGGSSQVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSSDHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMFFDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAKHEAASPSQTVQRAVSVNPGKADPSKLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNESLRRESVRPV 2e12-scFv-IgAH IgA-T4-CD80(nucleotide sequence) (SEQ ID NO: 181)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac 2e12-scFv-IgAH IgA-T4-CD80 (amino acid sequence) (SEQ IDNO: 182) MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESSGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESLTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLATGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDADPSNNLLPSWAITLISVNGIFVICCLTYCFAPRCRER RRNERLRRESVRPV2e12-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (nucleotide sequence) (SEQ IDNO: 183)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctataggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcactccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggacatggacgtggacttgtccaccgcctctaccacgcaggcgggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata2e12-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (amino acid sequence) (SEQ IDNO: 184) MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDHVDSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTTTCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKADPSKLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 500A2 scFv (nucleotide sequence) (SEQID NO: 185)atgttgtatacatctcagctccttgggcttttactcttctggatttcagcctccagaagtgacatagtgctgactcagactccagccactctgtctctaattcctggagaaagagtcacaatgacctgtaagaccagtcagaatattggcacaatcttacactggtatcaccaaaaaccaaaggaggctccaagggctctcatcaagtatgcttcgcagtccattcctgggatcccctccagattcagtggcagtggttcggaaacagatttcactctcagcatcaataacctggagcctgatgatatcggaatttattactgtcaacaaagtagaagctggcctgtcacgttcggtcctggcaccaagctggagataaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtcaagctgcagcagtccggttctgaactagggaaacctggggcctcagtgaaactgtcctgcaagacttcaggctacatattcacagatcactatatttcttgggtgaaacagaagcctggagaaagcctgcagtggataggaaatgtttatggtggaaatggtggtacaagctacaatcaaaaattccagggcaaggccacactgactgtagataaaatctctagcacagcctacatggaactcagcagcctgacatctgaggattctgccatctattactgtgcaagaaggccggtagcgacgggccatgctatggactactggggtcaggggatccaagttaccgtctcctctgatc 500A2 scFv (amino acid sequence) (SEQ ID NO: 186)MLYTSQLLGLLLFWISASRSDIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPSRFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIKRGGGGSGGGGSGGGGSQVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSYNQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTVSSD NT 5′ oligo: Name   :IgGWT3 (SEQID NO: 187) GTTGTTTTCGAAGGATCCGCTTTACCCGGAGACAGGGAGAGGCTCTT NT 3′ oligo:Name   :hIgGWT5 (SEQ ID NO: 188)GTTGTTAGATCTGGAGCCCAAATCTTGTGACAAAACTCACACATG NT 5′ oligo: Name   :FADD5(SEQ ID NO: 189) SequenceGTTGTGGATCCTTCGAACCCGTTCCTGGTGCTGCTGCACTCGGTGTCG NT 3′ oligo:Name   :FADD3 (SEQ ID NO: 190) SequenceGTTGTTATCGATCTCGAGTTATCAGGACGCTTCGGAGGTAGATGCGTC NT FADD-CSSCFV(nucleotide sequence) (SEQ ID NO: 191)gtggatccttcgaacccgttcctggtgctgctgcactcggtgtcgtccagcctgtcgagcagcgagctgaccgagctcaagttcctatgcctcgggcgcgtgggcaagcgcaagctggagcgcgtgcagagcggcctagacctcttctccatgctgctggagcagaacgacctggagcccgggcacaccgagctcctgcgcgagctgctcgcctccctgcggcgccacgacctgctgcggcgcgtcgacgacttcgaggcgggggcggcggccggggccgcgcctggggaagaagacctgtgtgcagcatttaacgtcatatgtgataatgtggggaaagattggagaaggctggctcgtcagctcaaagtctcagacaccaagatcgacagcatcgaggacagatacccccgcaacctgacagagcgtgtgcgggagtcactgagaatctggaagaacacagagaaggagaacgcaacagtggcccacctggtgggggctctcaggtcctgccagatgaacctggtggctgacctggtacaagaggttcagcaggcccgtgacctccagaacaggagtggggccatgtccccgatgtcatggaactcagacgcatctacctccgaagcgtcctgataactcgagatcgataacaacFADD-CSSCFV (amino acid sequence) (SEQ ID NO: 192)VDPSNPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMILLEQNDLEPGHTELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQLKVSDTKIDSIEDRYPRNLTERVRESLRIWKNTEKENATVAHLVGALRSCQMNLVADLVQEVQQARDLQNRSGAMSPMSWNSDASTSEAS HCD28tm5B (nucleotidesequence) (SEQ ID NO: 193)GTTGTGGATCCTCCCTTTTGGGTGCTGGTGGTGGTTGGTGTCCTGGCTTGCTAT AGCTTG HCD28tm3S(nucleotide sequence) (SEQ ID NO: 194)GTTGTYTCGAACCCAGAAAATAATAAAGGCCACTGTTACTAGCAAGCTATAGC AAGCCAG HCD28tm5′(nucleotide sequence) (SEQ ID NO: 195) GTTGTGGATCCTCCCTTTTGGGTGCTGGTGGTHCD28tm3′ (nucleotide sequence) (SEQ ID NO: 196)GTTGTTTCGAAGCCAGAAAATAATAAAGGCCAC HCD80tm5′ (nucleotide sequence) (SEQID NO: 197) GTTGTGGATCCTCCTGCTCCCATCCTGG HCD80tm3′ (nucleotide sequence)(SEQ ID NO: 198) GTTGTTTCGAACGGCAAAGCAGTAGGTCAGGC MFADD5BB (nucleotidesequence) (SEQ ID NO: 199) GTGTGGATCCTTCGAACCCATTCCTGGTGCTGCTGCACTCGCTGMFADD3XC (nucleotide sequence) (SEQ ID NO: 200)GTTGTTATCGATCTCGAGTGAGGGTGTTTCTGAGGAAGACAC Murine FADD nucleotidesequence (full length, but without flanking −Ig or transmembranesequences) (nucleotide sequence) (SEQ ID NO: 201)gtggatccttcgaacatggacccattcctggtgctgctgcactcgctgtccggcagcctgtcgggcaacgatctgatggagctcaagttcttgtgccgcgagcgcgtgagcaaacgaaagctggagcgcgtgcagagtggcctggacctgttcacggtgctgctggagcagaccgacctggagcgcgggcacaccgggctgctgcgcgagttgctggcctcgctgcgccgacacgatctactgcagcgcctggacgacttcgaggcggggacggcgaccgctgcgcccccgggggaggcagatctgcaggtggcatttgacattgtgtgtgacaatgtggggagagactggaaaagactggcccgcgagctgaaggtgtctgaggccaagatggatgggattgaggagaagtacccccgaagtctgagtgagcgggtaagggagagtctgaaagtctggaagaatgctgagaagaagaacgcctcggtggccggactggtcaaggcgctgcggacctgcaggctgaatctggtggctgacctggtggaagaagcccaggaatctgtgagcaagagtgagaatatgtccccagtactaagggattcaactgtgtcttcctcagaaacaccctgactcgagatcgat Murine FADD(amino acid sequence) (SEQ ID NO: 202)VDPSNMDPFLVLLHSLSGSLSGNDLMELKFLCRERVSKRKLERVQSGLDLFTVLLEQNGLERGHTGLLRELLASLRHDLLQRLDDFEAGTATAAPPGEADLQVAFDIVCDNVGRDWKRLARELKVSEAKMDGIEEKYPRSLSERVRESLKVWKNAEKKNASVAGLVKALRTCRLNLVADLVEEAQESVSKSENMSPVLRDSTVSSSETP MCASP3-5 (nucleotidesequence) (SEQ ID NO: 203)GTTGTGGATCCTTCGAACATGGAGAACAACAAAACCTCAGTGGATTCA MCASP3-3 (nucleotidesequence) (SEQ ID NO: 204) GTTGTTATCGATCTCGAGCTAGTGATAAAAGTACAGTTCTTTGGTMCASP8-5 (nucleotide sequence) (SEQ ID NO: 205)GTTGTTTCGAACATGGATTTCCAGAGTTGTCTTTATGCTATTGCTG MCASP8-3 (nucleotidesequence) (SEQ ID NO: 206)GTTGTTATCGATGTCGAGTCATTAGGGAGGGAAGAAGAGCTTCTTCCG hcasp3-5 (nucleotidesequence) (SEQ ID NO: 207) GTTGTGGATCCTTCCAACATGGAGAACACTGAAAACTCAGTGGAThcasp3-3 (nucleotide sequence) (SEQ ID NO: 208)GTTGTTATCGATCTCGAGTTAGTGATAAAAATAGAGTTCTTTTGTGAG hcasp8-5 (nucleotidesequence) (SEQ ID NO: 209) GTTGTGGATCCTTCGAACATGGACTTCAGCAGAAATCTTTATGAThcasp8-3 (nucleotide sequence) (SEQ ID NO: 210)GTTGTTATCGATGCATGCTCAATCAGAAGGGAAGACAAGTTTTTTTCT 1. 2H7 scFv withalternative VHL11 mutations (SEQ ID NO: 211): Nucleotide sequenceAagcttgccgccatggattttcaaggtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgag (one of the following: tcn, acn,gan, can, aan, cgn, agn)gtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggttaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagAmino acid sequence (SEQ ID NO: 212)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAE (one of the following: S, T,D, E, Q, N, R, K, H)VRPGASVKMSCKASGYIFTSYNMHWVKQTPRQGLEWIGAIYFGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVT VSSDQ 2. VHL11deletion (SEQ ID NO: 213) Nucleotide sequence:Aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgaggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactcgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO:214):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAEVRPGASVKMSCKASGYTFTSYNMHWVKQTFRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQ 3. 2H7 VL L106 withalternative mutations Nucleotide sequence (SEQ ID NO:215):aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggag (tcn, agn, aan, cgn, can, gan, andnon-natural derivatives of these codons)aaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctc Amino acid sequence(SEQ ID NO: 216):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLITISRVEAEDAATYYCQQWS FNPPTFGAGTKLE(S, T, R, K, H, Q, N, D, E, and non-natural derivatives of these aminoacids at position 106) KDGGGSGGGGSGGGGSS 4. VL L106 deletion (SEQ ID NO:217) Nucleotide sequence:Aagcttgccgccatggattttcaagagcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctc Amino acid sequence: (SEQ ID NO: 218)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLEKDGGGSGGGGSGGGGSS 5. IgE CH3 CH4 (SEQ ID NO: 219)Nucleotide sequence:tccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctagaaAmino acid sequence (SEQ ID NO: 220):SNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGTEYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK 6. hIgG1H/IgE WCH3WCH4 (SEQ ID NO:221) Nucleotide sequence:tgatcaggagcccaaatcttctgacaaaactcacacatcccaccgtccccagcatccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagcagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcacgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcccaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctagaa Amino acid sequence(SEQ ID NO:222):DQEPKSSDKTHTSPPSFASSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQT VQRAVSVNGK 7.IgE WCH2 WCH3 WCH4 (SEQ ID NO: 223) Nucleotide sequence:TgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctagaAmino acid sequence (SEQ ID NO: 224):DHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTWSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVDTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSNPGK 8. hIgG1H/IgE CH3 CH4(ORF) (SEQ ID NO: 225) Nucleotide sequence:tgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcatccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggaaaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatccttcgaa Amino acidsequence (SEQ ID NO: 226):DQEPKSSDKTHTSPPSPASNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPPVNHSTRKEFKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPKKTKGSGFFVFSRLEVTRAEWEGQKDEFICRAVHEAASPSQTVQRAVSVNPGKSGSFE 9. 2H7 VHL11S scFv hIgG1(SSS-S)H hIgE WCH3 WCH4 (SEQ IDNO: 227) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcatccaacccgagaggggtgagcgcctaccaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctagaAmino acid sequence (SEQ ID NO: 228):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYYPGNGDTSYNQKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSASNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNP GK 10. 2H7VHL11S scFv hIgG1(SSS-P)H hIgE WCH3 WCH4 (SEQ ID NO: 229) Nucleotidesequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggcaaagcagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcatccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggctcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgcccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtctgtaaatcccggtaaatgataacctagaAmino acid sequence (SEQ ID NO: 230):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSPASNPRGVAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFGRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNP GK 10. 2H7 VLL106S (SEQ ID NO: 231)aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctc Amino acid sequence (SEQ ID NO: 232):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLESKDGGGSGGGGSGGGGSS 11. 2H7 VL L106S scFv (SEQ ID NO: 233)Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagtctaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagtcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO: 234):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLESKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQ 12. 2H7 scFv VL L106SVHL11S scFv (SEQ ID NO: 235) Nucleotide sequence:Aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagtctaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO: 236):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLASKDGGGSGGGGSGGGGSSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQ 10. Human IgD hingelinker with attached restriction sites (SEQ ID NO: 237) Nucleotide:gtggatccaggttcgaagtctccaaaggcacaggcctcctccgtgcccactgcacaaccccaagcagagggcagcctcgccaaggcaaccacagccccagccaccacccgtaacacaggaagaggaggagaagagaagaagaaggagaaggagaaagaggaacaagaagagagagagacaaagaccggtgcagtcgacg Amino acid (SEQ ID NO: 238):VDPGSKSPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQ EERETKTGAVDSequence of Native IgD hinge domain (SEQ ID NO: 239): (includes acysteine residue-we truncated the hinge prior to that residue for theseconstructs:) Nucleotide:gagtctccaaaggcacaggcctcctccgtgcccactgcacaaccccaagcagagggcagcctcgccaaggcaaccacagccccagccaccacccgtaacacaggaagaggaggagaagagaagaagaaggagaaggagaaagaggaacaagaagagagagagacaaagacaccagagtgtccgagccacacccagcctcttggcgtctacctgctaacccct Amino acidsequence (SEQ ID NO: 240):ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTP 12. 2H7 VH L11S (SEQ ID NO: 241) Nucleotidesequence:caggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttct Amino acid sequence (SEQ ID NO: 242):QAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWY FDVWGTGTTVTVSS13. 2H7 VH L11S scFv (SEQ ID NO: 243) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctaaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttaactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO: 244):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDVKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQ 14. 2H7 scFv VH L11ShIgG1 (CSC-S)H WCH2 WCH3 (SEQ ID NO: 245) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtctgtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatctccaccgtgctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 246):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSCDKTHTSPPCSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 15. 2H7scFv VH L11S IgE WCH2 WCH3 WCH4 (SEQ ID NO: 247) Nucleotide sequence:aagctgccgccatggattttcaagagcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactccctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtctgtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggctaattctgtgcaagagtggtgtactatagtaactctcactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcacgtcgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctccgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctaga Amino acid sequence (SEQ ID NO: 248):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKCGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTALCLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK 16. 2H7 scFv VH L11S mIgE WCH2WCH3 WCH4 (SEQ ID NO: 249) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtctgtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatactgtgcaagagtggtgtactatagtaactcttactggtactcgatgtctggggcacagggaccacggtcaccgtctcttctgatcacgttcgacctgtcaacatcactgagcccaccttggagctactccattcatcctgcgaccccaatgcattccactccaccatccagctgtactgcttcatttatggccacatcctaaatgatgtctctgtcagctggctaatggacgatcgggagataactgatacacttgcacaaactgttctaatcaaggaggaaggcaaactagcctctacctgcagtaaactcaacatcactgagcagcaatggatgtctgaaagctccttcacctgcacggtcacctcccaaggcgtagactcttggcccacactcggagatgcccagatcatgagccacggggtgtgattacaacctgatcccacccagccccctggacctgtatcaaaacggtgctcccaagcttacctgtctggtggtggacctggaaagcgagaagaatgtcaatgtgacgtggaaccaagagaagaagacttcagcctcagcatcccagtggtacactaagcaccacaataacgccacaactagtatcacctccatcctgcctgtagttgccaaggactggattgaaggctacggctatcagtgcatagtggaccaccctgattttcccaagcccattgtgcgttccatcaccaagaccccaggccagcgctcagcccccgaggtatatgtgttcccaccaccagaggcggagagcgaggacaaacgcacactcacctgtttgatccagaacttcttccctgaggatatctctgtgcagtggctgggggatggcaaactgaactcaaacagccagcacagtaccacaacacccctgaaatccaatggctccaatcaaggcttcttcatcttcagtcgcctagaggtcgccaagacactctggacacagagaaaacagttcacctgccaagtgatccatgaggcacttcagaaacccaggaaactggagaaaacaatatccacaagccttggtaacacctccctccgtccatcctagtaatctagagAmino acid sequence (SEQ ID NO: 250):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDHVRPVNITEPTLELLHSSCDPNSFHSTIQLYCFIYGHILNDVSVSWLMDDREITDTLAQTVLIKEEGKLASTCSKLNITEQQWMSESTFTCKVTSQGVDYLAHTRRCPDHEPRGVITYLIPPSPLDLYQNGAPKLTCLVVDLESEKNVNVTWNQEKKTSVSASQWYTKHHNNATTSITSILPVVAKDWIEGYGYQCIVDHPDFPKPIVRSITKTPGQRSAPEVYVFPPPEEESEDKRTLTCLIQNFFPEDISVQWLGDGKLISNSQHSTTTPLKSNGSNQGFFIFSRLEVAKTLWTQRKQFTCQVIHEALQKPRKLEKTISTSLGNTSLRPS 17. 2H7 scFv VH L11S hIgA WHWCH2 T4CH3 (SEQ ID NO: 251) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtctgtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctaga Amino acid sequence (SEQ ID NO: 252):MDFQVQIFSFLLISASVIIARGQIVOLLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTTVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHPPALEDLLLGSSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLA GKPTHVNVSVVMAEVD18. 2H7 scFv VH L11S mIgA WH WCH2 T4 CH3 (SEQ ID NO: 253) Nucleotidesequence:aagcttgccgccatggatttcaagtgcagatttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatccccaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcaccatcagcagagtggaggctgaagatgctgccacttattaacgccagcagtggagttttaacccacccacgttcggagctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttacctacagcagtctggggctgagtctgtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcacatctgttctcctcctactactcctcctccaccttcctgccagcccagcctgtcactgcagcggccagctcttgaggacctgctcctgggttcagatgccagcatcacatgtactctgaatggcctgagagatcctgagggagctgtcttcacctgggagccctccactgggaaggatgcagtgcagaagaaagctgtgcagaattcctgcggctgctacagtgtgtccagcgtcctgcctggctgtgctgagcgctggaacagtggcgcatcattcaagtgcacagttacccatcctgagtctgacaccttaactggcacaattgccaaagtcacagtgaacaccttcccaccccaggtccacctgctaccgccgccgtcggaggagctggccctgaatgagctcgtgtccctgacatgcctggtgcgagctttcaaccctaaagaagtgctggtgcgatggctgcatggaaatgaggagctgtccccagaaagctacctagtgtttgagcccctaaaggagccaggcgagggagccaccacctacctggtgacaagcgtgttgcgtgtatacagctgaaatctggaaacagggtgaccagtactcctgcatggtgggccacgaggccttgcccatgaacttcacccagaagaccatcgaccgtctgtcgggtaaacccaccaatgtcagcgtgtctgtgatcatgtcagagggagattgataatctagatAmino acid sequence (SEQ ID NO: 254):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDHICSPPTTPPPPSCQPSLSLQRPALEDLLLGSDASITCTLNGLRDPEGAVFTWEPSTGKDAVQKKAVQNSCGCYSVSSVLPGCAERWNSGASFKCTVTHPESDTLTGTIAKVTVNTFPPQVHLLPPPSEELALNELVSLTCLVRAFNPKEVLVRWLHGNEELSPESYLVFEPLKEPGEGATTYLVTSVLRVSAEIWKQGDQYSCMVGHEALPMNFTQKTIDRLSGKPTNVSVS VIMSEGD A. mIgAWCH2 T4CH3 (SEQ ID NO: 255) Nucleotide sequence:Gttgttgatcacatctgttctcctcctactactcctcctccaccttcctgccagcccagcctgtcactgcagcggccagctcttgaggacctgctcctgggttcagatgccagcatcacatgtactctgaatggcctgagagatcctgagggagctgtcttcacctgggagccctccactgggaaggatgcagtgcagaagaaagctgtgcagaattcctgcggctgctacagtgtgtccagcgtcctgcctggctgtgctgagcgctggaacagtggcgcatcattcaagtgcacagttacccatcctgagtctgacaccttaactggcacaattgccaaagtcacagtgaacaccttcccaccccaggtccacctgctaccgccgccgtcggaggagctggccctgaatgagctcgtgtccctgacatgcctggtgcgagctttcaaccctaaagaagtgctggtgcgatggctgcatggaaatgaggagctgtccccagaaagctacctagtgtttgagcccctaaaggagccaggcgagggagccaccacctacctggtgacaagcgtgttgcgtgtatcagctgaaatctggaaacagggtgaccagtactcctgcatggtgggccacgaggccttgcccatgaacttcacccagaagaccatcgaccgtctgtcgggtaaacccaccaatgtcagcgtgtctgtgatcatgtcagagggagattgataatctagat Amino acidsequence (SEQ ID NO: 256):DHICSPPTTPPPPSCQPSESLQPPALEDLLLGSDASITCTLNGLRDPEGAVFTWEPSTGKDAVQKKAVQNSCGCYSVSSVLPGCAERWNSGASFKCTVTHPESDTLTGTIAKVTVNTFPPQVHLLPPPSEELALNELVSLTCLVRAFNPKEVLVRWLHGNEELSPESYLVFEPLKEPGEGATTYLVTSVLRVSAEIWKQGDQYSCMVGHEALPMNFTQTIDRL SGKPTNVSVSVIMSEGD20. K322S CH2 region (SEQ ID NO: 257) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgcagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgctcggtcaccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaaAmino acid sequence (SEQ ID NO: 258):PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCSVSNKALPAPIEKTISKAK 21. K322S CH2WCH3 (SEQ ID NO: 259) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgctcggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 260):PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCSVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK 1. K322L CH2 WCH3 (SEQID NO: 261) Nucleotide sequence:tgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcctggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ ID NO:262): DQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCLVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPYLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 22.2H7 scFv VHL11S hIgG1 (SSS-S)H K322SCH2 WCH3 (SEQ ID NO: 263) Nucleotidesequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggaggctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagactggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcccacaatcagaagttctagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgccatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttccccttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgtggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgctcggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 264):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCSVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFNYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 23. 2H7 scFvVHL11S hIgG1 (SSS-S)H K322LCH2 WCH3 (SEQ ID NO: 265) Nucleotidesequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcctggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccggtaaatgatctaga Amino acidsequence (SEQ ID NO: 266):MDFQVQIFSFLLISASVIIARGQIYLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVTFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCLVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 24. 2H7 scFvVHL11S hIgG1 (CSS-S)H K322SCH2 WCH3 (SEQ ID NO: 267) Nucleotidesequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctamctgtgcaagagtggtgtactatagtaactcttactggtacucgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgctcggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 268):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGITVTVSSDQEPKSCDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCSVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 25. P331SCH2 (SEQ ID NO: 269) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaaAmino acid sequence (SEQ ID NO: 270)PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKBYKCKVSNKALPASIEKTISKAK 26. P331S CH2WCH3 (SEQ ID NO: 271) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccoggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacottgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 272)PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVHEALHNHYTQKSLSLSPGK 27. 2H7 scFv VH L11S(SSS-S)H P331S CH2 WCH3 (SEQ ID NO: 273) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttcctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 274)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVENAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTNPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 28. 2H7 scFvVH L11S (CSS-S)H P331S CH2 WCH3 (SEQ ID NO: 275) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 276)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYSNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSCDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGPYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 29. T256NCH2 region (SEQ ID NO: 277) Nucleotide sequence:CctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggaaccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaaAmino acid sequence (SEQ ID NO: 278)PELLGGPSVFLFPPKPKDTEMISRNPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 30. T256N CH2WCH3 (SEQ ID NO: 279) Nucleotide sequence:cctgaactcctggggggaccgtcagtcctcctcttccccccaaaacccaaggacaccctcatgatctcccggaaccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctacaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 280)PELLGGPSVFLFPPKPKDTLMISRNPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 31. 2H7 scFv VH L11S(SSS-S)H T256N CH2 WCH3 (SEQ ID NO: 281) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccaaactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgattaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggaaccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 282)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVVEAEDAATYYCQQWSFNPPTHGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRNPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 32. 2H7 scFvVH L11S (CSS-S)H T256N CH2 WCH3 (SEQ ID NO: 283) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaatgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggaaccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 284)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSCDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRNPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKETVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 33.RTPE/QNAK (255-258) CH2 (SEQ ID NO: 285) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccagaacgctaaggtcacatgcgtggtggggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaaAmino acid sequence (SEQ ID NO: 286)PELLGGPSVFLFPPKPKDTLMISQNAKVTCVVVDVSHEDPEXTKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 34. RTPE/QNAK(255-258)CH2 WCH3 (SEQ ID NO: 287) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccagaacgctaaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 288)PELLGGPSVFLFPPKPKDTLMISQNAKVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKGKVSNKALPALPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 35. 2H7 scFv VH L11S(SSS-S)H RTPE/QNAK (255-258)CH2 WCH3 (SEQ ID NO: 289) Nucleotidesequence:aagctgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccagaacgctaaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagcttaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 290)MDFQVQIFSFLLISASVIIARGQIVLSQPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTKGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISQNAKTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 36. 2H7scFv VH L11S (CSS-S)H RTPE/QNAK (255-258)CH2 WCH3 (SEQ ID NO: 291)Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacattaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaagctcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccagaacgctaaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgccaaatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaattgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagatgagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 292)MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSCDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISQNAKVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 36. K290QCH2 region (SEQ ID NO: 293) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagacccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacacagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaaAmino acid sequence (SEQ ID NO: 294):PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 37. K290Q CH2WCH3 (SEQ ID NO: 295) Nucleotide sequence:CctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacacagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccccgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 296):PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSGEDPEVKFNWYVDGVEVHNAKTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 38. 2H7 scFv VH L11S(SSS-S)H K290Q CH2 WCH3 (SEQ ID NO: 297) Nucleotide sequence:aagcttgcccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggaaaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctattccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacacagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 298):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 39. 2H7 scfvVH L11S (CSS-S)H K290Q CH2 WCH3 (SEQ ID NO: 299) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggctctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctaacaatcagcagagtggaggctgaagatgctgaagatgctgccactcattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatcaacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggaactatttatccaggaaatggtgatacttcctacaatcagaagtcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacacagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 300):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMIHWYQQKPGSSPKPW1YAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMIIWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFGARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSCDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHBDPEVKFNWYVDGVEVHNAKTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKGKVSNKALPAJ4EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 40. A339PCH2(SEQ ID NO: 301) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaacccaaaAmino acid sequence (SEQ ID NO: 302):PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPK 41. A339P CH2WCH3 (SEQ ID NO: 303) Nucleotide sequence:cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaacccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccccgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggccccgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 304):PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKLPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHTEALHNHYTQKSLSLSPGK 42. 2H7 scFv VHL11S(SSS-S)H A339P CH2 WCH3 (SEQ ID NO: 305) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgocaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaacccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 306):MSFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLFLLKDGGGSGGGGSGGGGSSQAYLQQSGAEVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYQMLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 43. 2H7 scFvVHL11S (CSS-S)H A339P CH2 WCH3 (SEQ ID NO: 307) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgccgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccactcattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgacgatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcccagcctacatgcagctcagcagcctgacatctgaagactctgaggtctatttctgtgcaagagtggtgtactatagtaactcttactggcacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaacccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 308):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSVDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 44. G28-1VH(SEQ ID NO: 309) Nucleotide sequence:gcggtccagctgcagcagtctggacctgagctggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO: 310):AVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLIVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWG QGTSVTVSSDQ 45.G28-1VL (SEQ ID NO: 311) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgcgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtca Amino acid sequence (SEQ ID NO: 312):MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPMDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSS 46. G28-1 scFv (SEQ ID NO: 313)Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatatgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgatacagttatttggcttggtatcagcagaaacagggaaaatcacatcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagctggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactgggtcaaggaacctcagtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO: 314):MVSTAQFLGLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQ 47. G28-1 VHL11S (SEQ ID NO:315) Nucleotide sequence:gcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggctttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO: 316):AVQLQQSGPESEKPGASVKISCKASGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQ GTSVTVSSDQ 48.G28-1 VHL11S scFv (SEQ ID NO: 317) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctcgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggccctatggactggggtcaaggaacctcagtcaccgtctcttctgatcag Amino acid sequence (SEQ ID NO: 318):MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQ 49. G28-1 scFv (SSS-S)H WCH2WCH3 (SEQ ID NO: 319) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttattttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagctggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcatgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgCcaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ IDNO: 320): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPELEKLPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDHDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 50. G28-1 scFvIgAW H IgG1WCH2 WCH3 (SEQ ID NO: 321) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagctggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgcgcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 322):MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQKHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQPVPSTPPTPSPSTPPTPSPSCAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 51. G28-1 scFvVHL11S (SSS-S)H WCH2 WCH3 (SEQ ID NO: 323) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaaggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatcccaccgtcctcagcacctgaactctgggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ ID NO:324): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDHDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 52. G28-1 scFvVHL11S (CSS-S)H WCH2 WCH3 (SEQ ID NO: 325) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggcyccyycyyccycyacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ ID NO:326): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKPOLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQEPKSCDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 53. G28-1 scFv VHL11S (CSC-S)H WCH2 WCH3 (SEQ ID NO: 327) Nucleotide sequence:aagcttcccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggcaatgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacaftgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacactctccaccgtgctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgsggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ ID NO:328): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQEPKSCDKTHTSPPCSAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 54. G28-1 scFv VHL11S (SSC-P)H WCH2 WCH3 (SEQ ID NO: 329) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacagggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcgcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaaggagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactggggtcaaggaacctcagtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagacctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgcctggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence: (SEQ ID NO:330): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIEKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNDPYYGGTTYRKFKATLTVDKSSST.AYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQEPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK II. 54. HCTLA4 HIGG1(SSS-S)H P238SCH2 WCH3 (SEQ ID NO: 331) Nucleotide sequence:atggcttgccttggatttcagcggcacaaggctcagctgaacctggctgccaggacctggccctgcactctcctgtttttcttctcttcatccctgtcttctgcaaagcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgacggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaatgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctcgatcaacccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctcctcagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacgcataatgccaagacaaagccgcggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaAmino acid sequence (SEQ ID NO: 332):MACLGFQRHKAQLNLAARTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMTGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQPKSSDKTHTSPPSSAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 55. Fc2-2VL (SEQ ID NO: 333) Nucleotide sequence:gttgttaagcttgccgccatggattcacaggcccaggttcttatgttactgctgctatgggtatctggtacctgtggggacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggtttctatgagctgcaagtccagcagagccttttatataatcacaatcaaaagaactacttggcctggtaccagcagataccagggcagtctcctaaactgctgattttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcagagtgaaggctgaagacctggcagtttattactgtcagcaatattatacctatcctcccacgttcggaggtggcaccaagctggaaataaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctcg Amino acid sequence (SEQ ID NO: 334):MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVSMSCKSSQSLLYNHNQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVKAEDLAVYYCQQYYTYPPTFGGGTKLEIKGGGGSGGGGSGGGGSS 56. FC2-2VH (SEQ ID NO: 335)Nucleotide sequence:Gggagctcgcaggtgcagttgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccgtctatggtgttaactgggttcgccagcctccaggaaagggtctggactggctgggaatgatatggggatggaagcacagactataattcagctcaaatccactgagcatcagtaaggacaactccaagagccaagttttcttaaaaatggacagtctacaaactgatgacacagccaggtactactgtgccagagatcactatggtacccactatgctatggactactggggcaaggaacctcagtcaccgtctcctctgatcag Amino acid sequence (SEQ ID NO: 336):GSSQVQLKESGPGLVAPSQSLSITCTVSGFSLTVYGVNWVRQPPGKGLDWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMDSLQTDDTARYYCARDHYGTHYAM DYWGQGTSVTVSSDQ57. FC2-2scFv (SEQ ID NO: 337) Nucleotide sequence:gttgttaagcttgccgccatggattcacaggcccaggttcttatgttactgctgctatgggtatctggtacctgtggggacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggtttctatgagctgcaagtccagtcagagccttttatataatcacaatcaaaagaactacttggcctggtaccagcagataccagggcagtctcctaaactgctgatttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcagagtgaaggctgaagacctggcagtttattactgtcagcaatattatacctatcctcccacgttcggaggtggcaccaagctggaaataaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctctcaggtgcagttgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccgtctatggtgttaactgggttcgccagcctccaggaaagggtctggactggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcagtaaggacaactccaagagccaagttttcttaaaaatggacagtctacaaactgatgacacagccaggtactactgtgccagagatcactatggtacccactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcag Amino acid sequence (SEQ IDNO: 338): MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVSMSCKSSQSLLYNHNQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVKAEDLAVYYCQQYYTYPPTFGGGTKLEIKGGGGSGGGGSGGGGSSQVQLKESGPGLVAPYSQSLSITCTVSGFSLTVYGVNWVRQPPGKGLDWLGMIWGDGSTDYNSALKSLSISKDNSKSQVFLKMDSLQTDDTARYYCARDHYAMDYWGQGTSVTVVSSDQ 58. FC2-2 VHL11S (SEQ IDNO: 339) Nucleotide sequence:gggagctctcaggtgcagttgaaggagtcaggacctggctcggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccgtctatggtgttaactgggttcgccagcctccaggaaagggtctggactggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcagtaaggacaactccaagagccaagttttcttaaaaatggacagtctacaaactgatgacacagccaggtactactgtgccagagatcactatggtacccactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcag Amino acid sequence (SEQ ID NO: 340):(GSS)QVQLKESGPGSVAPSQSLSITCTVSGFSLTVYGVNWVRQPPGKGLDWLGMIWGDGSTDYNSALKSRLSISKDNSKSSQVFLKMDSLQTDDTARYYCARDHYGTHYA MDYWGQGTSVTVSSDQ59. FC2-2 VH L11S scFv (SEQ ID NO: 341) Nucleotide sequence:gttgttaagcttgccgccatggattcacaggcccaggttcttatgttactgctgctatgggtatctggtacctgtggggacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggtttctatgagctgcaagtccagtcagagccttttatataatcacaatcaaaagaactacttggcctggtaccagcagataccagggcagtctcctaaactgctgatttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcagagtgaaggctgaagacctggcagtttattactgtcagcaatattatacctatcctcccacgttcggaggtggcaccaagctggaaataaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctctcaggtgcagttgaaggagtcaggacctggctcggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccgtctatggtgttaactgggttcgccagcctccaggaaagggtctggactggctgggaatgatatggggtgatggaagcacactataattcagctcaaatccagactgagcatcagtaaggacaactccaagagccaagttttcttaaaaatggacagtctacaaactgatgacacagccaggtactactgtgccagagatcactatggtacccactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcag Amino acid sequence (SEQ IDNO: 342): MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVSMSCKSSQSLLYNHNQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVKAEDLAVYYCQQYYTYPPTFGGGTKLEIKGGGGSGGGGSGGGGSSQVQLKESGPGSVAPSQSLSITCTVSGFSLTVYGVNWVRQPPGKGLDWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMDSLQTDDTARYYCARDHYGTHYAMDYWGQGTSVTVSSDQ 60. FC2-2 (SSS-S)HWCH2 WCH3 (SEQ ID NO: 343) Nucleotide sequence:gttgttaagcttgccgccatggattcacaggcccaggttcttatgttactgctgctatgggtatctggtacctgtggggacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggtttctatgagctgcaagtccagtcagagccttttatataatcacaatcaaaagaactacttggcctggtaccagcagataccagggcagtctcctaaactgatttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcagagtgaagctgaagacctggcagtttattactgtcagcaatattatacctatcctcccacgttcggaggtggcaccaagctggaaataaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctctcaggtgcagttgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccgtctatggtgttaactgggttcgccagcctccaggaaagggtctggactggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcagtaaggacaactccaagagccaagttttcttaaaaatggacagtctacaaactgatgacacagccaggtactactgtgccagagatcactatggtacccactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 344):MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVSMSCKSSQSLLYNHNQKNYLAWYQQIPGQSPKLLIYWASTRIESGVPDRFTGSGSGTDFTLTISRVKAEDLAVYYCQQYYTYPPTFGGGTKLEIKGGGGSGGGGSGGGGSSQVQLKESGPGLVAPSQSLSITCTVSGFSLTVYGVNWVRQPPGKGLDWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMDSLQTDDTARYYCARDHYGTHYAMDYWGQGTSVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 61.FC2-2 VHL11S (SSS-S)H WCH2 WCH3 (SEQ ID NO: 345) Nucleotide sequence:gttgttaagcttgccgccatggattcacaggcccaggttcttatgttactgctgctatgggtatctggtacctgtggggacattgtgatgtcacagtctccatcctccctagctgtgtcagttggagagaaggtttctatgagctgcaagtccagtcagagccttttatataatcacaatcaaaagaactacttggcctggtaccagcagataccagggcagtctcctaaactgctgatttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcagagtgaaggctgaagacctggcagtttattactgtcagcaatattatacctatcctcccacgttcggaggtggcaccaagctggaaataaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctctcaggtgcagttgaaggagtcaggacctggctcggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccgtctatggtgttaactgggttcgccagcctccaggaaagggtctggactggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcagtaaggacaactccaagagccaagttttcttaaaaatggacagtctacaaactgatgacacagccaggtactactgtgccagagatcactatggtacccactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 346):MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVSMSCKSSQSLLYNHNQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVKAEDLAVYYCQQYYTYPPTFGGGTKLEIIKGGGGSGGGGSGGGGSSQVQLKESGPGSVAPSQSLSITCTVSGFSLTVYGVNWVRQPPGKGLDWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMDSLQTDDTARYYCARDHYGTHYAMDYWGQGTSVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHTNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 62.UCHL-1 VH (SEQ ID NO: 347) Nucleotide sequence:atgggcaggcttacttcttcattcctgctactgattcctgcatatgtcctctcccagattactctgaaagagtctggccctgggatcttgcagccctcccagaccctcagtctgacttctttctctgggttttcactgaccacttatggtataggagtaggttggattcgtcagcctccagggaagggtctggagtggctgacacacatttggtggaatgataataagtactataacacagccctgaggagccggctcacaatctccaaggattcctccaacaaccaagtactcctcaagatcgccaatgtggacactgcagataccgccacatactactgtctctacggctacacttactggggccaagggactctggtcactgtctctgca Amino acid sequence (SEQ IDNO: 348): MGRLTSSFLLLIVPAYVLSQITLKESGPGILQPSQTLSLTCSFSGFSLTTYGIGVGWIRQPPGKGLEWLTHIWWNDNKYYNTALRSRLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGTLVTVSA 63. UCHL-1 VL (SEQ ID NO: 349) Nucleotide sequence:atgaagttgcctgcctgttaggctgttggtgctgatgttctggattcctgcttccatcagtgatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttcttttacagtaatggaaacacctatttacattggtacctgcagaagcccaggccagtctccaaaactcctgatctacaaacttttccaaccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaa Amino acid sequence (SEQ ID NO:350): MKLPVRLLVLMFWIPASISDVVMTQTPLSLPVSLGDQASISCRSSQSLLYSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK 64. UCHL-1 scFv (SEQ ID NO: 351) Nucleotide sequence:gttgttaagcttgccgccatgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttccatcagtgatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttctttacagtaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaaactcctgatctacaaactttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcagattactctgaaagagtctggccctgggatcttgcagccctcccagaccctcagtctgacttgttctttctctgggttttcactgaccacttatggtataggagtaggttggattcgtcagcctccagggaagggtctggagtggctgacacacatttggtggaatgataataagtactataacacagccctgaggagccggctcacaatctccaaggattcctccaacaaccaagtactcctcaagatcgccaatgtggacactgcagataccgccacatactactgtctctacggctacacttactggggccaagggactctggtcactgtctctgctgatca Amino acid sequence (SEQ ID NO: 352):MKLPVRLLVLMFWIPASISDVVMTQTPLSLPVSLGDQASISCRSSQSLLYSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKDGGGSGGGGSGGGGSSQITLKESGPGILQPSQTLSLTCSFSGFSLTTYGIGVGWIRQPPGKGLEWLTHIWWNDNKYYNTALRSRLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGTLVTVSAD 65. UCHL-1 VH I11SL12S (SEQ ID NO:353) Nucleotide sequence:gggagctctcagattactctgaaagagtctggccctgggatcttgcagccctcccagaccctcagtcgacttgttctttctctgggttttcactgaccacttatggtataggagtaggttggattcgtcagcctccagggaagggtctggagtggctgacacacatttggtggaatgataataagtactataacacagccctgaggagccggctcacaatctccaaggattcctccaacaaccaagtactcctcaagatcgccaatgtggacactgcagataccgccacatactactgtctctacggctacacttactggggccaagggactctggtcactgtctctgctgatca Amino acid sequence: (SEQ ID NO: 354)(GSS)QITLKESGPGSSQPSQTLSLTCSFSGFSLTTYGIGVGWIRQPPGKGLEWLTHIWWNDNKYYNTALRSRLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGT LVTVSAD 66.UCHL-1 scFv VH L11S (SEQ ID NO: 355) Nucleotide sequence:gttgttaagcttgccgccatgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttccatcagtgatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttctttacagtaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaaactcctgatctacaaactttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcagattactctgaaagagtctggccctgggagctcccagccctcccagaccctcagtctgacttgttctttctctgggttttcactgaccacttatggtataggagtaggttggattcgtcagcctccagggaagggtctggagtggctgacacacatttggtggaatgataataagtactataacacagccctgaggagccggctcacaatctccaaggattcctccaacaaccaagtactcctcaagatcgccaatgtggacactgcagataccgccacatactactgtctctacggctacacttactggggccaagggactctggtcactgtctctgctgatca Amino acid sequence (SEQ ID NO: 356):MKLPVRLLVLMFWIPASISDVVMTQTPLSLPVSLGDQASISCRSSQSLLYSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEADLGVYFCSQSTHVPWTFGGGTKLEIKDGGGSGGGGSGGGGSSQITLKESGPGSSQPSQTLSLTCSFSGFSLTTYGIGVGWIRQPPGKGLEWLTHIWWNDNKYYNTALRSRLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGTLVTVSAD 67. UCHL-1 scFv (SSS-S)H WCH2 WCH3(SEQ ID NO: 357) Nucleotide sequence:gttgttaagcttgccgccatgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttccatcagtgatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttctttacagtaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaaactcctgatctacaaactttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcagattactctgaaagagtctggccctgggatcttgcagccctcccagaccctcagtctgacttgttctttctctgggttttcactgaccacttatggtataggagtaggttggattcgtcagcctccagggaagggtctggagtggctgacacacatttggtggaatgataataagtactataacacagccctgaggagccggctcacaatctccaaggattcctccaacaaccaagtactcctcaagatcgccaatgtggacactgcagataccgccacatactactgtctctacggctacacttactggggccaagggactctggtcactgtctctgctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ IDNO: 358): MKLPVRLLVLMFWIPASISDVVMTQTPLSLPVSLGDQASISCRSSQSLLYSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKDGGGSGGGSGGGGSSQITLKESGPGILQPSQTLSLTCSFSGFSLTTYGIGVGWIRGPPGKGLEWLTHIWWNDNKYYNTALRSLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGTLVTVSADQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 68. UCHL-1 scFv VHL11S(SSS-S)H WCH2 WCH3 (SEQ ID NO: 359) Nucleotide sequence:gttgttaagcttgccgccatgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttccatcagtgatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttctttacagtaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaaactcctgatctacaaactttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcagattactctgaaagagtctggccctgggagctcccagccctcccagaccctcagtctgacttgttctttctctgggttttcactgaccacttatggtataggagtaggttggattcgtcagcctccagggaagggtctggagtggctgacacacatttggtggaatgataataagtactataacacagccctgaggagccggctcacaatctccaaggattcctccaacaaccaagtactcctcaagatcgccaatgtggacactgcagataccgccacatactactgtctctacggctacacttactggggccaagggactctggtcactgtctctgctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaa Amino acid sequence (SEQID NO: 360): MKLPVRLLVLMFWIPASISDVVMTQTPLSLPVSLGDQASISCRSSQSLLYSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTKLISRVEAEDLGVYFCSQSTHPWTFGGGTKLEIKDGGGSGGGGSGGGGSSQITLKESGPGSSQPSQTLSLTCSFSGFSLTTYGIGVGWIRQPPGKGLEWLTHIWWNDNKYYNTALRSRLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGTLVTVSADQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK 69. 5B9 VH L11S (SEQ IDNO: 361) Nucleotide sequence:gggagctctcaggtgcagctgaagcagtcaggacctggctcagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcag Amino acid sequence (SEQ ID NO: 362):(GSS)QVQLKQSGPGSVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYY YAMDYWGQGTSVTVSS73. 5B9 VH L11S scFv (SEQ ID NO: 363) Nucleotide sequence:aggcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctctcaggtgcagctgaagcagtcaggacctggctcagtgcagtcctcacagsgcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcag Amino acid sequence (SEQ ID NO:364): MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGSVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSS 70. 5B9 scFv VIIL11S(SSS-S)H WCH2 WCH3 (SEQ ID NO: 365) Nucleotide sequence:aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggtggcggtggctcgggcggtggtggtgggtcgggtggcggcgggagctctcaggtgcagctgaagcagtcaggacctggctcagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagagAmino acid sequence (SEQ ID NO: 366):MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPGSVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDQVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 76. 2H7scFv VH L11S (SSS-S)H P238SCH2 WCH3 (SEQ ID NO: 367) Nucleotidesequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtcgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacucctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagcacagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggcaccgctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 368):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 78. 2H7 scFvVR L11S (SSS-S)H WCH2 WCH3 (SEQ ID NO: 369) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacacccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctcccttctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 370):MDFQVQIFSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHIWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 79. 2H7 scFvVH L11S (CSS-S)H WCH2 WCH3 (SEQ ID NO: 371) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 372):MDFQVQIFSFSLLISFLLISASVIIARGQUVLSQSPAILSPAILSASPGEKVYMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDQEPKSCDDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNAKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEALHNHYTQKSLSLS PGK 77. G8-1 scFvVHL11S (SCS-S)H WCH2 WCH3 (SEQ ID NO: 373) Nucleotide sequence:gttgttaagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactggggtcaaggaacctcsgtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatgcccaccgtcctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag˜cagcctgacctgcctg˜c˜aggcftctatccaagcgacatcgcc˜ggcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagag Amino acid sequence (SEQID NO: 374): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKSSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQEPKSSDKTHTCPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKWYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 79.. G28-1 scFvVHL11S (CCS-P)H WCH2 WCH3 (SEQ ID NO: 375) Nucleotide sequence:gttgttaagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatgtccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ IDNO: 376): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQEPKSCDKTHTCPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 80. G28-1 scFv VHL11S (SCC-P)H WCH2 WCH3 (SEQ ID NO: 377) Nucleotide sequence:gttgttaagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ IDNO: 378): MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKPQLLVSFAKTLAGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQKHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEALHNHYTQKSLSPGK 81. G28-1 scFv VH L11SmIgE CH2 CH3 CH4 (SEQ ID NO: 379) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcacgttcgacctgtcaacatcactgagcccaccttggagctactccattcatcctgcgaccccaatgcattccactccaccatccagctgtactgcttcatttatggccacatcctaaatgatgtctctgtcagctggctaatggacgatcgggagataactgatacacttgcacaaactgttctaatcaaggaggaaggcaaactagcctctacctgcagtaaactcaacatcactgagcagcaatggatgtctgaaagcaccttcacctgcaaggtcacctcccaaggcgtagactatttggcccacactcggagatgcccagatcatgagccacggggtgtgattacctacctgatcccacccagccccctggacctgtatcaaaacggtgctcccaagcttacctgtctggtggtggacctggaaagcgagaagaatgtcaatgtgacgtggaaccaagagaagaagacttcagtctcagcatcccagtggtacactaagcaccacaataacgccacaactagtatcacctccatcctgcctgtagttgccaaggactggattgaaggctacggctatcagtgcatagtggaccaccctgattttcccaagcccattgtgcgttccatcaccaagaccccaggccagcgctcagcccccgaggtatatgtgttcccaccaccagaggaggagagcgaggacaaacgcacactcacctgtttgatccagaacttccctgaggatatctgtgcaggctgggggatggcaaactgatctcaaacagccagcacagtaccacaacacccctgaaatccaatggctccaatcaaggcttcatcttcagtcgcctagaggtcgccaagacactctggacacagagaaaacagttcacctgccaagtgatccatgaggcacttcagaaacccaggaaactggagaaaacaatatccacaagccttggtaacacctccctccgtccatcctagaatctagagg Aminoacid sequence (SEQ ID NO: 380):MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDHVRPVNITEPTLELLHSSCDPNAFHSTIQLYCFIYGHILNDVSWLMDDREITDTLAQTVLIKEEGKLASTCSKLNITEQQWMSESTFTCKVTSQGVDYLAHTRRCPDHEPRGVITYLIPPSPLDLYQNGAPKLTCLVVDLESEKNVNVTWNQEKKTSVSASQWYTKAHHNNATTSITSILPVVAKDWIEGYGYQCIVDHPDFPKPIVRSITKTPGQRSAPEVYVFPPPEEESEDKRTLTCLIQNFFPEDISVQWLGDGKLISNSQHSTTTPLKSNGSNQGFFIFSRLEVAKTLWTQRKQFTCQVIHEALQKPRKLEKTISTSLGNTSLRPS 82. G28-1 scFv VH L11S mIgA WH WCH2T4CH3 (SEQ ID NO: 381) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgssgstttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcacatctgttctcctcctactactcctcctccaccttcctgccagcccagcctgtcactgcagcggccagctcttgaggacctgctcctgggttcagatgccagcatcacatgtactctgaatggcctgagagatcctgagggagctgtcttcacctgggagccctccactgggaaggatgcagtgcagaagaaagctgtgcagaattcctgcggctgctacagtgtgtccagcgtcctgcctggctgtgctgagcgctggaacagtggcgcatcattcaagtgcacagttacccatcctgagtctgacaccttaactggcacaagccaaagtcacagtgaacaccttcccaccccaggtccacctgctaccgccgccgtcggaggagctggccctgaatgagctcgtgtccctgacatgcctggtgcgagctttcaaccctaaagaagtgctggtgcggatggctgcatggaaatgaggagctgtccccagaaagctacctagtgtttgagcccctaaaggagccaggcgagggagccaccacctacctggtgacaagcgtgttgcgtgtatcagctgaaatctggaaacagggtgaccagtactcctgcatggtgggccacgaggccttgcccatgaacttcacccagaagaccatcgaccgtctgtcgggtaaacccaccaatgtcagcgctgtgatcatgtcagagggagattgataatctagat Amino acid sequence(SEQ ID NO: 382):MVSTAQFLGLLLLWLTGGRGCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDHICSPPPPPSCQPSLSLQRPALEDLLLGSDASITCTLNGLRDPEGAVFTWEPSTGKDAVQKKAVQNSCSGCYSVSSVLPGCAERWNSGASFKCTVTHPESDTLTGTIAKVTVNTFPPQVHLLPPPSEELALNELVSLTCLVRAFNPKEVLVRWLHGNEELSPESYLVFEPLKEPGEGATTYLVTSVLRVSAEIWKQGDQYSCMVGHEALPMNFTQKTIDRLSGKPTNVSVSVIMSEGD 83. G28-1 scFvVh L11S hIgE CH2 CH3 CH4 (SEQ ID NO: 383) Nucleotide sequence:aagcttgccgccatggtatccacagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcgggcacttcccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaatgataatctagaAmino acid sequence (SEQ ID NO: 384):MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDHVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQHTFEDSTKKCADSNPRGVSAYLSRPSPEDLFIRKSPTITCLVVDLAPSKGTVNLWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQTVQRAVSVNPGK 84. G28-1 scFv VH L11S hIgA WH WCH2T4CH3 (SEQ ID NO: 385) Nucleotide sequence:atggtatccacagctcagctcagttccttgggttgctgctgctgtggcttacaggtggcagatgtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcagcggtccagctgcagcagtctggacctgagtcggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcttctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccaccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgcctcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgcccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctagaAmino acid sequence (SEQ ID NO: 386):MVSTAQFLGLLLLWLTGGRCDIQMTQSPASLSASVGETVTITCRTSENVYTSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSSAVQLQQSGPESEKPGASVKISCKASGYSFTGYNMNVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALED 85. HD37 VL (SEQ ID NO: 387) Nucleotide sequence:aagcttgccgccatggagacagacacactcctgctatgggtgctgctgctctgggttccaggctccactggtgacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggaggcaccaagctggaaatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctcg Amino acid sequence (SEQ ID NO: 388):METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQPATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSS 86. HD37 VH (SEQ ID NO: 389)Nucleotide sequence:gggagctcgcaggttcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcag Amino acid sequence (SEQ IDNO: 390): (GSS)QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSDQ 87. HD37 scFv (SEQ ID NO: 391) Nucleotidesequence:aagcttgccgccatggagacagacacactcctgctatgggtgctgctgctctgggttccaggctccactggtgacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcacctcaacatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggaggcaccaagctggaaatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggtcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcag Amino acid sequence(SEQ ID NO: 329):METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSQSVDYDGDSYLNYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIIKGGGGSGGGGSGGGGSSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSDQ 88. HD37 VHL11S(SEQ ID NO: 393): Nucleotide sequence:gggagctcgcaggttcagctgcagcagtctggggctgagtcggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcag Amino acid sequence (SEQ IDNO: 394): (GSS)QVQLQQSGAESVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSDQ 89. HD37 scFv VHL11S (SEQ ID NO: 395): Nucleotidesequence:Aagcttgccgccatggagacagacacactcctgctatgggtgctgctgctctgggttccaggctccactggtgacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccaccagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggaggcaccaagctggaaatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctcgcaggttcagctgcagcagtctggggctgagtcggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcag Amino acidsequence (SEQ ID NO: 396):METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSSQVQLQQSGAESVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRTTTVGRYYYAMDYWGQGTSVTIVSSDQ 90. HD37 scFv IgAHhIgG1 WCH2 T4CH3 (SEQ ID NO: 397) Nucleotide sequenceaagcttgccgccatggagacagacacactcctgctatgggtgctgctgctctgggttccaggctccactggtgacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaagggatgctgcaacctatcactgcagcaaagtactgaggatccgtggacgttcggtggaggcaccaagctggaaatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctcgcaggttcagctgcagcagtctggggctgagtcggtgaggcctgggtccctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatccccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctaga Amino acid sequence (SEQID NO: 398) METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSSQVQLQQSGAESVRPGSSVKLSCKASGYAFSSYWNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVD 91. HD37 scFv (SSS-S)H WCH2 WCH3 (SEQ ID NO: 399)Nucleotide sequence:aagcttgccgccatggagacagacacactcctgctatgggtgctgctgctctgggttccaggctccactggtgacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggaggcaccaagctggaaatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatcgtcacaggttcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence: (SEQ ID NO: 400)METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKISLSLSPGK 92.HD37 scFv VH L11S (SSS-S)H WCH2 WCH3 (SEQ ID NO: 401) Nucleotidesequence:aagcttgccgccatggagacagacacactcctgctatgggtgctgctgctctgggttccaggctccactggtgacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggaggcaccaagctggaaatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcgggagctcgcaggttcagctgcagcagtctggggctgagtcggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaagccaaagggcagccccgagaaccacaggtgtacacctgcccccatcccgggatgagctgaccaagaaccaactacaagacccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 402):METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSSQVQLQQSGAESVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 91. L6VL (SEQ ID NO: 403) Nucleotide sequence:atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacattgacttgcagggccagctcaagtgtaagtttcatgaactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaatttggcttctgagttccctggtcgcttcagtggcgagtggtctgggacctcttactctctcgcaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggaatagtaaccactcacgttcggtgctgggaccaagctggagctgaaagagctctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct Amino acid sequence (SEQ ID NO: 404):MDFQVQIFSFLLISASVIMSRGQIVLSQSPAILSASPGEKVTLTCRASSSVSFMNWTQQKPGSSPKPWIYATSNLASEFPGRFSGEWSGTSYSLAISRVEAEDAATYYCQQWNSNPLTFGAGTKLELKELSGGGGSGGGGSGGGGGS 92. L6 VH (SEQ ID NO: 405) Nucleotidesequence:cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggacagccaacatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgcctacactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagatttagctatggtaactcacgttacgctgactactggggccaaggcaccactctcacagtctcctctgatca Amino acid sequence (SEQ ID NO: 406):QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGQPTYADDFKGRFAFSLETSAYTAYLQINNLKNEDMATYFCARFSYGNSRYADY WGQGTTLTVSSD 93.L6 scFv (SEQ ID NO: 407) Nucleotide sequence:atggattttcaagtgcagattttcagxttcctgctaatcagtgcttcagtcataatgtccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacattgacttgcagggccagctcaagtgtaagtttcatgaactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaatttggcttctgagttccctggtcgcttcagtggcgagtggtctgggacctcttactctctcgcaatcagcagagtggaggctgaagatgctgccacacttattactgccagcagtggaatagtaacccactcacgttcggtgctgggaccaagctggagctgaaagagctctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctctgcagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggacagccaacatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgcctacactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagatttagctatggtaactaactcacttacgctgactactggggccaaggcaccactctcacagtctcctctgatca Amino acid sequence (SEQ ID NO: 408):MDFQVQIFSFLLISASVIMSRGQIVLSQSPAILSASPGEKVTLTCRASSSVSFMNWYQQKPGSSPKPWIYATSNLASEFPGRFSGEWSGTSYSLAISRVEAEDAATYYCQQWNSNPLTFGAGTKLELKELSGGGGSGGGGSGGGGSLQIQLVGSGPELKKPGETVKISCKASGYTFNYGMNWVKQAPGKGLKWMGWINTYTGQPTYADDFKGRFAFSLETSAYTAYLQINNLKNEDMATYFCARFSYGNSRYADYWGQGTTLTVSSD 94. L6 VHL11S (SEQ ID NO:409) Nucleotide sequence:ctgcagatccagttggtgcagtctggacctgagtcgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggacagccaacaatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgcctacactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagatttagctatggtaactcacgttacgctgactactggggccaaggcaccactctcacagtctcctctgatca Amino acid sequence (SEQ ID NO: 410):QIQLVQSGPESKKPGETVKISCKASGYTPTNYGMNWVKQAPGKGLKWMGWINTYTGQPTYADDFKGRFAFSLETSAYTAYLQINNLKNEDMATYFCARFSYGNSRYADY WGQGTTLTVSSD 95.L6 VH L11S scFv (SEQ ID NO: 411) Nucleotide sequence;Aagcttgttgttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacattgacttgcagggccagctcaagtgtaagtttcatgaactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaatttggcttctgagttccctggtcgcttcagtggcgagtggtctgggacctcttactctctcgcaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggaatagtaacccactcacgttcggtgctgggaccaagctggagctgaaagagctctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctctgcagatccagttggtgcagtctggacctgagtcgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggacagccaacatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgcctacactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagatttagctatggtaactcacgttacgctgactactggggccaaggcaccactctcacagtctcctctgatca Amino acid sequence (SEQ ID NO: 412):MDFQVQTFSFLLISASVIMSRGQIVLSQSPAILSASPGEKVTLTCRASSSVSFMNWYQQQKPGSSPKPWIYATSNLASEFPGRFSGEWSGTSYSLAISRVEADAATYYCQQWNSNPLTFGAGTKLELKELSGGGGSGGGGSGGGGSLQIQLVQSGPESKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGQPTYADDFKGRAFAFSLETSAYTAYLQINNLKNEDMATYFCARFSYGNSRYADYWGQGTTLTVSSD 96. L6 Or L6 VHL11S scFvIgAH hIgG1 WCH2 WCH3 (SEQ ID NO: 413) Nucleotide sequence: (L6 VHL11S isis shown)aagcttgttgttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggacaaattgttctctcccagtctccagcaatcctgtcgcatctccaggggagaaggtcacattgacttgcagggccagctcaagtgtaagtttcatgaactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaatttggcttctgagttccctggtcgcttcagtggcgagtggtctgggacctcttactctctcgcaatcagcagcagagtggaggctgaagatgctgccacttattactgccagcagtggaatagtaacccactcacgttcggtgctgggaccaagctggagctgaaagagctctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctctgcagatccagttggtgcagtctggacctgagtcgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggacagccaacatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgcctacactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagatttagctatggtaactcacgttacgctgactactggggccaaggcaccactctcacagtctcctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgcgcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagatttagctatggtaactcacgttacgctgactactggggccaaggcaccactctcacagtctcctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgcgcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagacctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtgcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccacracacgcagaagagcctctccctgtctccgggtaaatgatctagaAmino acid sequence (SEQ ID NO: 414):MSFQVQIFSFFLLISASVIMSRGQIVLSQSPAILSASPGEKVTLTCRASSSVSFMNWYQQKPGSSPKPWIYATSNLASEFPGRFSGEWSGTSYSLAISRVEAEDAATYYCQQWNSNPLTFGAGTKLELKELSGGGGSGGGGSGGGGSLQIQLVQSGPESKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYGQPTYADDFKGRFAFSLETSAYTAYLQINNLKNEDMATYFCARFSYGNSRYWGQGTTLTVSSDQPVPSTPPTPSPSTPPTPSPSCAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEALHNHYTQKSL SLSPGK 97. L6scFv VHL11S (SSS-S)H WCH2 WCH3 (SEQ ID NO: 415) Nucleotide sequence:aagcttgttgttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacattgacttgcagggccagctcaagtgtaagtttcatgaactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaatttggcttctgagttccctggtcgcttcagtggcgagtggtctgggacctcttactctctcgcaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggaatagtaacccactcacgttcggtgctgggaccaagctggagctgaaagagctctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctctgcagatccagttggtgcagtctggacctgagtcgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggacagccaacatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgcctacactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagatttagctatggtaactcacgttacgctgactactggggccaaggcaccactctcacagtctcctctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acidsequence (SEQ ID NO: 416):MDFQVQIFSFLLISASVIMSRGQIVLSQSPAILSASPGEKVTLTCRASSSVSFMNYQQKPGSSPKPWIYATSNLASEFPGRFSGEWSGTSYSLAISRVEAEDAATYYCQQWNSNPLTFGAGTKLELKELSGGGGSGGGGSGGGGSLQIQLVQSGPESKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGQPTYADDFKGRFAFSLETSAYTAYLQINNLKNEDMATFCARFSYGNSRYADYWGQGTTLTVSSDQEPKSSDKTHTSPPSSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 98. P238 CH2WCH3 a. P238S CH2 WCH3 (SEQ ID NO: 417) Nucleotide sequence:cctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcacgtcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggaaatgatctagaAmino acid sequence (SEQ ID NO: 418):PELLGGSSVFLFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK b. (SSS-S)H P238S CH2WCH3 (SEQ ID NO: 419) Nucleotide sequence:tgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ ID NO:420): DQEPKSSDKTHTSPPSSAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPENVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK*99. a.CD16-6 low (ED) + NL + (SSS-S)H P238S CH2 WCH3 (SEQ ID NO: 421)Nucleotide sequence:ggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggqtcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga Amino acid sequence (SEQ ID NO: 422):MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLADQEPKSSDKTHTSPPSSAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPKIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSSVMEALHNHYTQKSLSLSGK 100. b.CD16-6 low (ED) +HE4LP + (SSS-S)H P238S CH2 WCH3 (SEQ ID NO: 423) Nucleotide sequence:aagcttgccgccatgcctgcttgtcgcctaggcccgctagccgccgccctcctcctcagcctgctgctgttcggcttcaccctagtctcaggcaccggtgcaatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagttgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggcttgttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagccgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggaggagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaaaAmino acid sequence (SEQ ID NO: 424):MPACRLGPLAAALLLSLLLFGFTLVSGTGAMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLADQEPKSSDKTHTSPPSSAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFYLSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 101. CD16-9 high(ED) (SSS-S)H P238S CH2 CH3 a. CD16-9 high (ED)NL + (SSS-S)H P238S CH2CH3 (SEQ ID NO: 425) Nucleotide sequence:gttgttaagcttgccgccatgtggcagctgctcctcccaactgctctgctacttctagtttcagctggcatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggcttttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaaa Amino acid sequence (SEQ ID NO: 426):MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLADQEPKSSDKTHTSPPSSAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CD16-9 high (ED) + HE4LP +hIgG1 (SSS-S)H P238S CH2 CH3 (SEQ ID NO: 427) Nucleotide sequence:aagcttgccgccatgcctgcttgtcgcctaggcccgctagccgccgccctcctcctcagcctgctgctgttcggcttcaccctagtctcaggcaccggtgcaatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgaccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatattacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggctttttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggctgatcaggagcccaaatcttctgacaaaactcacacatccccaccgtcctcagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggaagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctagaaa Aminoacid sequence (SEQ ID NO: 428):MPACRLGPLAAALLLSLLLFGFTLVSGTGAMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLADQEPKSSDKTHTSPPSSAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 102. a. CD16 EDlow (native leader) (SEQ ID NO: 429) Nucleotide sequence:aagcttgccgccatgtggcagctgctcctcccaactgctctgctacttctagtttcagctggcatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagacggctcctacttctgcagggggcttgttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggctgatcaaaaAmino acid sequence (SEQ ID NO: 430):mwqlllptallllvsagmrtedlpkavvflepqwyrvlekdsvtlkcqgayspednstqwfhneslissqassyfidaatvddsgeyrcqtnlstlsdpvqlevhigwlllqaprwvfkeedpihlrchswkntalhkvtylqngkggrkyfhhnsdfyipkatlkdsgsyfcrglvgsknvssetvnititqgladq b. CD16 ED low (HE4 leader) (SEQ ID NO:431) Nucleotide sequenceaagcttgccgccatgcttgtcgcctaggcccgctagccgccgccctcctcctcagcctgctgctgttcggcttcaccctagtctcaggcaccggtgcaatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggcttgttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggctgatcaaa Amino acid sequence (SEQ ID NO:432)mpacrlgplaaalllslllfgftlvsgtgamrtedpkavvflepqwyrvelekdsvtlkcqgayspednstqwfhneslissqassyfidaatvddsgeyrcqtnlstlsdpvqlevhigwlllqaprwvfkeedpihlrchswkntalhkvtylqngkgrkyfhhnsdfyipkatlkdsgsyfcrglvgsknvssetvnititqgladq 103. a. CD16 ED high (nativeleader) (SEQ ID NO: 433) Nucleotide sequence:gttgttaagcttgccgccatgtggcagctgctcctcccaactgctctgctacttctagtttcagctggcatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacaggactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggctttttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggctgatcaaa Amino acid sequence (SEQ ID NO: 434):mwqlllptallllvsgmrtedlpkavvflepqwyrvlekdsvtlkcqgayspednstqwfhneslissqassyfidaatvddsgeyrcqtnlstlsdpvqlevhigwlllqaprwvfkeedpihlrchswkntalhkvtylqngkgrkyfhhnsdfyipkatlkdsgsyfcrglfgsknvssetvnititqgladq b. CD16 ED high (HE4 leader) (SEQ ID NO:435) Nucleotide sequence:aagcttgccgccatgcctgcttgtcgcctaggcccgctagccgccgccctcctcctcagcctgctgctgttcggcttcaccctagtctcaggcaccggtgcaatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggctttttgggagtaaaaatgtgtcucagagactgtgaacatcaccatcactcaaggtttggctgatcaaa Amino acid sequence (SEQ ID NO:436): MPACRLGPLAAALLLSLLLFGFTLVSGTGAMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLADQ 104. 2e12 scFv (SSS-S)HP238S CH2 WCH3-hCD80TM/CT (SEQ ID NO: 437) Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggftagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat Amino acid sequence(SEQ ID NO: 438):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEHNVRNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNTKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 105. 10A8 scFv(SSS-S)H P238SCH2 WCH3-hCD80TM/CT (SEQ ID NO: 439) Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagccaggcatcccatcaaggttcagggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaacttattattgtcaacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatggatgggccacataagccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactactggggtcaaggaacctcagtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat Amino acid sequence (SEQ ID NO: 440):MDFQVQIFSFLLISASVIMSRGVDIQMTQSPSSLSASLGGKVTITCKASQDIKKYIGWYQHKPGKGPRLLLYYTSTLQPGIPSRFSGSGSGRDYSLSLIRNLEPDIATYYCQQYDNLPLTFGSGTKLEIKRGGGGSGGGGSGGGGSDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGFYWNWIRQFPGNKLEWMGHISHDGRNNYNPSLINRISITRDTSKNQFFLKLSSVTTEDTATYFCARHYGSSGAMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYGFAPRCRERRRNERLRRESVRPV 106. 2H7 scFv VHL11S(SSS-P)H P238SCH2CH3-hCD80TM/CT (SEQ ID NO: 441) Nucleotide sequence:aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaattggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctcttctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatcctcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat Amino acid sequence (SEQ ID NO:442): MDFQVQSFLLISASVIIARGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAESVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 107. G19-4 scFv(SSS-P)H P238SCH2 WCH3-hCD80TM/CT (SEQ ID NO: 443) Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgtaaactcctgatctactacacatcaagatacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgggagctcggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctatcgatgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgtcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacaagcccaccgagcccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatactcgag Amino acidsequence (SEQ ID NO: 444):MDFQVQIFSFLLISASVIMSRGVDIQMTQTTSSLSASLGDRVTISCRASQDIRNYNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRELGGGGSGGGGSGGGGSIDEVQLQQSGPELVKPGASMSCKASGYSFTGYIVNWLKQSHGKNLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSCFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVR PV 108. 2e12scFv IgA WH WCH2 T4 CH3-hCD80TM/CT (SEQ ID NO: 445) Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagccrgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacacaattctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac Amino acid sequence (SEQ ID NO: 446):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDADPSNNLLPSWAITLISVNGIFVICCLTYCFAPRCRER RRNERLRRESVRPV109. 2e12 scFV (SSS-P)H P238S CH2 WCH3-mFADD-TM/CT (SEQ ID NO: 447)Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacccattcctggtgctgctgcactcgctgtccggcagcctgtcgggcaacgatctgatggagctcaagttcttgtgccgcgagcgcgtgagcaaacgaaagctggagcgcgtgcagagtggcctggacctgttcacggtgctgctggagcagaacgacctggagcgcgggcacaccgggctgctgcgcgagttgctggcctcgctgcgccgacacgatctactgcagcgcctggacgacttcgaggcggggacggcgaccgctgcgcccccgggggaggcagatctgcaggtggcatttgacattgtgtgtgacaatgtggggagagactggaaaagactggcccgcgagctgaaggtgtctgaggccaagatggatgggattgaggagaagtacccccgaagtctgagtgagcgggtaagggagagtctgaaagtctggaagaatgctgagaagaagaacgcctcggtggccggactggtcaaggcgctgcggacctgcaggctgaatctggtggctgacctggtggaagaagcccaggaatctgtgagcaagagtgagaatatgtccccagtactaagggattcaactgtgtcttcctcagaaacaccctgactcgagatcgat Amino acid sequence (SEQ ID NO: 448):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATICRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDPFLVLLHSLSGSLSGNDLMELKFLCRERVSKRKLERVQWSGLDLFTVLLEQNDLERGHTGLLRELLASLRRHDLLQRLDDFEAGTATAAPPPGEADLQVAFDIVCDNVGRDWKRLARELKVSEAKMDGIEEKYPRSLSERVRESLKVWKNAEKKNASVAGLVKALRTCRLNLVADLVEEAQESVSKSENMSPVLRDSTVSSSETP 110. 2e12 scFv(SSS-P)H WCH2WCH3-mFADD-TM/CT (SEQ ID NO: 449) Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactacttgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacccattcctggtgctgctgcactcgctgtccggcagcctgtcgggcaacgatctgatggagctcaagttcttgtgccgcgagcgcgtgagcaaacgaaagctggagcgcgtgcagagtggcctggacctgttcacggtgctgctggagcagaacgacctggagcgcgggcacaccgggctgctgcgcgagttgctggcctcgctgcgccgacacgatctactgcagcgcctggacgacttcgaggcggggacggcgaccgctgcgcccccgggggaggcagatctgcaggtggcatttgacattgtgtgtgacaatgtggggagagactggaaaagactggcccgcgagctgaaggtgtctgaggccaagatggatgggattgaggagaagtacccccgaagtctgagtgagcgggtaagggagagtctgaaagtctggaagaatgctgagaagaagaacgcctcggtggccggactggtcaaggcgctgcggacctgcaggctgaatctggtggctgacctggtggaagaagcccaggaatctgtgagcaagagtgagaatatgtccccagtactaagggattcaactgtgtcttcctcagaaacaccctgactcgagatcgat Amino acid sequence (SEQ ID NO:450): MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKYKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDPSNMDPFLVLLHSLSGSLSGNDLMELKYLCRERVSKLERVQSGLDLFTVLLEQNDLERGHTGLLRELLASLRRHDLLQRLDDFEAGTATAAPPGEADLQVAFDIVCDNVGRDWKRLARELKVSEAKMDGIEEKYPRSLSERVRESLKVWKNAEKKNASVAGLVKALRTCRLNLVADLVIEEAQESVSKSENMSPVLRDSTVSSSETP 111. mcasp3-TM/CT(SEQ ID NO: 451) Nucleotide sequence:GgatccttcgaacatggagaacaacaaaacctcagtggattcaaaatccattaataattttgaagtaaagaccatacatgggagcaagtcagtggactctgggatctatctggacagtagttacaaaatggattatcctgaaatgggcatatgcataataattaataataagaacttccataagagcactggaatgtcatctcgctctggtacggatgtggacgcagccaacctcagagagacattcatgggcctgaaataccaagtcaggaataaaaatgatcttactcgtgaagacattttggaattaatggatagtgtttctaaggaagatcatagcaaaaggagcagctttgtgtgtgtgattctaagccatggtgatgaaggggtcatttatgggacaaatgggcctgttgaactgaaaaagttgactagcttcttcagaggcgactactgccggagtctgactggaaagccgaaactcttcatcattcaggcctgccggggtacggagctggactgtggcattgagacagacagtgggactgatgaggagatggcttgccagaagataccggtggaggctgacttcctgtatgcttactctacagcacctggttactattcctggagaaattcaaaggacgggtcgtggttcatccagtccctttgcagcatgctgaagctgtacgcgcacaagctagaatttatgcacattctcactcgcgttaacaggaaggtggcaacggaattcgagtccttctccctggactccactttccacgcaaagaaacagatcccgtgtattgtgtccatgctcacgaaagaactgtacttttatcactagctcgagatcgatgAmino acid sequence (SEQ ID NO: 452):DPSNMENNKTSVDSKSINNFEVKTIHGSKSVDSGIYLDSSYKMDYPEMGICIIINNKNFHKSTGMSSRSGTDVDAANLRETFMGLKYQVRNKNDLTREDILELMDSVSKEDHSKRSSFVCVILSHGDEGVIYGTNGPVELKKLSFFRGDYCRSLTGKPKLFIIQACRGTELDCGIETDSGTDEEMACQKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCSMLKLYAHKLEFMHILTRVNRKVATEFESFSLDSTFHAKKQIPCIVSMLTKELYF YH 112. 2e12scFv (SSS-P)H WCH2WCH3-mcasp3-TM/CT (SEQ ID NO: 453) Nucleotidesequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtctccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctcggggtccctgccaggtttagtggcagtgggtctggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggagaacaacaaacctcagtggattcaaaatccattaataattttgaagtaaagaccatacatgggagcaagtcagtggactctgggatctatctggacagtagttacaaaatggattatcctgaaatgggcatatgcataataattaataataagaacttccataagagcactggaatgtcatctcgctctggtacggatgtggacgcagccaacctcagagagacattcatgggcctgaaataccaagtcaggaataaaaatgatcttactcgtgaagacattttggaattaatggatagtgtttctaaggaagatcatagcaaaaggagcagctttgtgtgtgtgattctaagccatggtgatgaaggggtcattttatgggacaaatgggcctgttgaactgaaaaagttgactagcttcttcagaggcgactactgccggagtctgactggaaagccgaaactcttcatcattcaggcctgccggggtacggagctggactgtggcattgagacagacagtgggactgatgaggagatggcttgccagaagataccggtggaggctgacttcctgtatgcttactctacagcagcacctggttactattcctggagaaattcaaaggacgggtcgtggttcatccagtccctttgcagcatgctgaagctgtacgcgcacaagctagaatttatgcacattctcactcgcgttaacaggaaggtggcaacggaattcgagtccttctccctggactccactttccacgcaaagaaacagatcccgtgtattgtgtccatgctcacgaaagaactgtacttttatcactagctcgagatcgatg Amino acid sequence(SEQ ID NO: 454):MDFQVQIFSFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMBSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMENNKTSVDSKSINNFEVKTIHGSKSVDSGIYLDSSYKMDYPEMDICIIINNKNFHKSTGMSSRSGTDVDAANLRETFMGLKYQVRNKNDLTREDILELMDSVSKEDHSKRSSFVCVILSHGDEGVIYGTNGPVELKKLTSFFRGDYCRSLTGKPKLFIIQACRGTELDCGIETDSGTDEEMACQKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCSMLKLYAHKLEFMHILTRVNRKVATEFESFSLDSTFHAKKQIPCIVSMLT KELYFYH 113.2e12 scFv (SSS-P)H P238SCH2WCH3-mcasp3-TM/CT (SEQ ID NO: 455) Nucleotidesequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacaxgcagaagagcctctccctgtctccgggtaaagcggatccttcgaattcgaacatggagaacaacaaaacctcagtggattcaaaatccattaataattttgaagtaaagaccatacatgggagcaagtcagtggactctgggatctatctggacagtagttacaaaatggattatcctgaaatgggcatatgcataataattaataataagaacttccataagagcactggaatgtcatctcgctctggttacggatgtggacgcagccaacctcagagagacattcatgggcctgaaataccaagtccaggaataaaaatgatcttactcgtgaagacattttggaattaatggatagtgtttctaaggaagatcatagcaaaaggagcagctttgtgtgtgtgattctaagccatggtgatgaaggggtcatttatgggacaaatgggcctgttgaactgaaaaagttgactagcttcttcagaggcgcatactgccggagtctgactggaaagccgaaactcttcatcattcaggcctgccggggtacggagctggactgtggcattgagacagacagtgggactgatgaggagatggcttgccagaagataccggtggaggctgacttcctgtatgcttactctacagcacctggttactattcctggagaaattcaaaggacgggtcgtggttcatccagtccctttgcagcatgctgaagctgtacgcgcacaagctagaatttatgcacattctcactcgcgttaacaggaaggtggcaacggaattcgagtccttctccctggactccactttccacgcaaagaaacagatcccgtgtattgtgtccatgctcacgaaagaactgtacttttatcactagctcgagatcgatcgatga Amino acidsequence (SEQ ID NO: 456):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYMGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNSNMENNKTSVDSKSINNFEVKTIHGSKSVDSGIYLDSSYKMDYPEMGICIIINNKNFHKSTGMSSRSGTDVDAANLRETFMGLKYQVRNKNDLTREDILELMDSVSKEDHSKRSSFVCVILSHGDEGVIYGTNGPVELKKLTSFFRGDYCRSLTGKPKLFIQACRGTELDCGIETDSGTDEEMACQKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCSMLKLYAHKLEFMHILTRVNRKVATEFESFSLDSTFHAKKQIPCIVSM LTKELYFYH 114.mcasp8-TM/CT (SEQ ID NO: 457) Nucleotide sequence:ttcgaacatggatttccagagttgtctttatgctattgctgaagaactgggcagtgaagacctggctgccctcaagttcctgtgcttggactacatcccacacaagaagcaggagaccatcgaggatgcccagaagctatttctgaggctgcgggaaaaggggatgttggaggaaggcaatctgtctttcctgaaagagctgcttttccacatcagtcggtgggacctgctggtcaacttcctagactgcaaccgagaggagatggtgagagagctgcgggatccagacaatgcccagatttctccctacagggtcatgctctttaagctctcagaagaagtgagcgagttggaattgagatcttttaagttccttttgaacaatgagatccccaaatgtaagctggaagatgacttgagcctgcttgaaatttttgtagaaatggagaagaggaccatgctggcagaaaataacttggaaaccctaaaatcaatctgtgaccaggtcaacaagagcctgctggggaagatcgaggattatgaaagatcaagcacagagagaagaatgagccttgaaggaagggaagagttgccaccttcagttttggatgagatgagcctcaaaatggcggaactgtgtgactcgccaagagaacaagacagtgagtcacggacttcagacaaagtttaccaaatgaagaacaaacctcggggatactgtctgatcatcaacaatcatgatttcagcaaggcccgggaagacataacccaactccgaaaaatgaaggacagaaaaggaacagactgtgataaagaggctctgagtaagacctttaaggagcttcattttgagatagtatcttacgacgactgcactgcaaatgaaatccacgagattctagaaggctaccaaagcgcagaccacaagaacaaagactgcttcatctgctgtatcctatcccacggtgacaagggtgtcgtctatggaacggatgggaaggaggcctccatctatgacctgacatcttacttcactggttcaaagtgcccttccctgtctgggaaacccaagatcttttcattcaggcttgccaaggaagtaacttccagaaaggagtgcctgatgaggcaggcttcgagcaacagaaccacactttagaagtggattcatcatctcacaagaactatattccggatgaggcagactttctgctgggaatggctacggtgaagaactgcgtttcctaccgagatcctgtgaatggaacctggtatattcagtcactttgccagagcctgagggaaagatgtcctcaaggagatgacattcttagcatcctgactggcgtgaactatgacgtgagcaataaagacgacaggaggaacaagggaaagcagatgccacagcccaccttcacactacggaagaagctcttcttccctccctaatgactcgagatcgattAmino acid sequence (SEQ ID NO: 458):SNMDFQSCLYAIAEELGSEDLAALKFLCLDYIPHKKQETIEDAQKLFLRLREKGMLEEGNLSFLKELLFHISRWDLLVNTNFLDCREEMVRELRDPDNAQISPYRVMLFKLSEEVSELELRSFKFLLNEIPKCKLEDDLSLLEIFVEMEKRTMLAENNLETLKSICDQVNKSLLGKIEDYERSSTERRMSLEGREELPPSVLDEMSLKMAELCDSPREQDSESRTSDKVYQMKNKPRGYCLIINNHDFSKAREDITQLRKMKDRKGTDCDKEALSKTFKELHFEIVSYDDCTANEIHEILEGYQSADHKNKDCFICCILSHGDKGVVYGTDGKEASIYDLTSYFTGSKCPSLSGKPKIFFIQACQGSNFQKGVPDEAGFEQQNHTLEVDSSSHKNYIPDEADFLLGMATVKNCVSYRDPVNGTWYIQSLCQSLRERCPQGDDILSILTGVNYDVSNKDDRRNKGKQMPQPTFTLRKKLFFPP 115. 2e12 scFv hIgG1 (SSS-P)H WCH2WCH3-mcasp8-TM/CT (SEQ ID NO: 459) Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggatttccagagttgtctttatgctattgctgaagaactgggcagtgaagacctggctgccctcaagttcctgtgcttggactacatcccacacaagaagcaggagaccatcgaggatgcccagaagctatttctgaggctgcgggaaaaggggatgttggaggaaggcaatctgtctttcctgaaagagctgcttttccacatcagtcggtgggacctgctggtcaacttcctagactgcaaccgagaggagatggtgagagagctgcgggatccagacaatgcccagatttctccctacagggtcatgctctttaagctctcagaagaagtgagcgagttggaattgagatcttttaagttccttttgaacaatgagatccccaaatgtaagctggaagatgacttgagcctgcttgaaattttgtagaaatggagaagaggaccatgctggcagaaaataacttggaaaccctaaaatcaatctgtgaccaggtcaacaagagcctgctggggaagatcgaggattatgaaaagatcaagcacagagagaagaatgagccttgaaggaagggaagagttgccaccttcagttttggatgagatgagcctcaaaatggcggactgtgtgactcgccaagagaacaagacagtgagtcacggacttcagacaaaagtttaccaaatgaagaacaaacctcggggatactgtctgatcatcaacaatcatgatttcagcaaggcccgggaagacataacccaactccgaaaaatgaaggacagaaaaggaacagactgtgataaagaggctctgagtaagaccttaaggaagcttcattttgagatagtatcttacgacgactgcactgcaaatgaaatccacgagattctagaaggctaccaaagcgcagaccacaagaacaaagactgcttcatctgctgtatcctatcccacggtgacaagggtgtcgtctatggaacggatgggaaggaggcctccatctatgacctgacatcttacttcactggttcaaagtgcccttccctgtctgggaaacccaagatctttttcattcaggcttgccaaggaagtaacttccagaaaggagtgcctgatgaggcaggcttcgagcaacagaaccacactttagaagtggattcatcatctcacaagaactatattccggatgaggcagactttctgctgggaatggctacggtgaagaactgcgtttcctaccgagatcctgtgaatggaacctggtatattcagtcactttgccagagcctgagggaaagatgtcctcaaggagatgacattcttagcatcctgactggcgtgaactatgacgtgagcaatggggacgacaggaggaacaagggaagcagatgccacagcccaccttcacactacggaagaagctcttcttccctccctaatgactcgagatcgattAmino acid sequence (SEQ ID NO: 460):MDFQVQIFSFLLISASVIMSRGVDIVLTTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKLPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNTKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDFQSCLYAIAEELGSEDLAALKFLCLDYIPHKKQETIEDAQKLFLRLREKGMLEEGNLSFLKELLFHISRWDLLVNFLDCNREEMVRELRDNAQISPYRVMLFKLSEEVSELELRSFKFLLNNEIPKCKLEDDLSLLEIFVEMEKRTMLAENNLETLKSICDQVNKSLLGKIEDYERSSTERRMSLEGREELPPSVLDEMSLKMAELCDSPREQDSESRTSDKVYQMKNKPRGYCLIINNHDFSKAREDITQLRKMKDRKGTDCDKEALSKTFKELHFEIVSYDDCTANEIHEILEGYQSADHKNKDCFICCILSHGDKGVVYGTDGKEASIYDLTSYFTGSKCPSLSGKPKIFFIQACQGSNFQKGVPDEAGFEQQNHTLEVDSSSHKNYIPDEADFLLGMATVKNCVSYRDPVNGTWYIQSLCQSLRERCPQGDDILSILTGVNYDVSNKDDRRNKGKQMPQPTFTLRKKLFFPP 116. 2e12 scFv hIgG1 (SSS-P)HP238SCH2 WCH3-mcasp8-TM/CT (SEQ ID NO: 461) Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcctggtggcgcccgcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggatttccagagttgtctttatgctattgctgaagaactgggcagtgaagacctggctgccctcaagttcctgtgcttggactacatcccacacaagaagcaggagaccatcgaggatgcccagaagctatttctgaggctgcgggaaaaggggatgttggaggaaggcaatctgtctttcctgaaagagctgcttttccacatcagtcggtgggacctgctggtcaacttcctagactgcaaccgagaggagatggtgagagagctgcgggatccagacaatgcccagatttctccctacagggtcatgctctttaagctctcagaagaagtgagcgagttggaattgagatcttttaagttccttttgaacaatgagatccccaaatgtaagctggaagatgacttgagcctgcttgaaatttttgtagaaatggagaagaggaccatgctggcagaaaataacttggaaaccctaaaatcaatctgtgaccaggtcaacaagagcctgctggggaagatcgaggattatgaaagatcaagcacagagagaagaatgagccttgaaggaagggaagagttgccaccttcagttttggatgagatgagcctcaaaatggcggaactgtgtgactcgccaagagaacaagacagtgagtcacggacttcagacaaagtttaccacctgaagaacaaacctcggggatactgtctgatcatcaacaatcatgatttcagcaaggcccgggaagacataacccaactccgaaaaatgaaggacagaaaaggaacagactgtgataaagaggctctgagtaagacctttaaggagcttcattttgagatagtatcttacgacgactgcactgcaaatgaaatccacgagattctagaaggctaccaaagcgcagaccacaagaacaaagactgcttcatctgctgtatcctatcccacggtgacaagggtgtcgtctatggaacggatgggaaggaggcctccatctatgacctgacatcttacttcactggttcaaagtgcccttccctgtctgggaaacccaagatctttttcattcaggcttgccaaggaagtaacttccagaaaggagtgcctgatgaggcaggcttcgagcaacagaaccacactttagaagtggattcatcatctcacaagaactatattccggatgaggcagactttctgctgggaatggctacggtgaagaactgcgtttcctaccgagatcctgtgaatggaacctggtatattcagtcactttgccagagcctgagggaaagatgtcctcaaggagatgacattcttagcatcctgactggcgtgaactatgacgtgagcaataaagacgacaggaggaacaagggaaagcagatgccacagcccaccttcacactacggaagaagctcttcttccctccctaatgactcgagatcgattcAmino acid sequence (SEQ ID NO: 462):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVGWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGNIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDFQSCLYAIAEELGSEDLAALKFLCLDYIPHKKQETIEDAQKLFLRLREKGMLEEGNLSFLKELLFHISRWDLLVNFLDCNREEMVRELRDPDNAQISPYRVMLFKLSEEVSELELRSFKFLLNNEIPKCKLEDDLSLLEIFVEMEKRTMLAENNLETLKSICDQVNKSLLGKIEDYERSSTERRMSLEGREELPPSVLDEMSLKMAELCDSPREQDSESRTSDKVYQMKNKPRGYCLIINNHDFSKAREDITQLRKMKDRKGTDCDKEALSKTFKELHFEIVSYDDCTANEIHEILEGYQSADHKNKDCFICCILSHGDKGVVYGTDGKEASIYDLTSYFTGSKCPSLSGKPKIFFIQACQGSNFQKGVPDEAGFEQQNHTLEVDSSSHYPDEADFLLGMATVKNCVSYRDPVNGTWYIQSLCQSLCQSLRERCPQGDDILSILTGVNYDVSNKDDRRNKGKQMPQPTFTLRKKLFFPP 117. hcasp3-TM/CT (SEQ ID NO:463) Nucleotide sequence:Ggatccttcgaacatggagaacactgaaaactcagtggattcaaaatccattaaaaatttggaaccaaagatcatacatggaagcgaatcaatggactctggaatatccctggacaacagttataaaatggattatcctgagatgggtttatgtataataattaataataagaattttcataaaagcactggaatgacatctcggtctggtacagatgtcgatgcagcaaacctcagggaaacattcagaaaacttgaaatatgaagtcaggaataaaaatgatcttacacgtgaagaaattgtggaattgatgcgtgatgtttctaaagaagatcacagcaaaaggagcagttttgtttgtgtgcttctgagccatggtgaaggaataatttttggaacaaatggacctgttgacctgaaaaaaataacaaactttttcagaggggatcgttgtagaagtctaactggaaaacccaaacttttcattattcaggcctgccgtggtacagaactggactgtggcattgagacagacagtggtgttgatgatgacatggcgtgtcataaaataccagtggaccgacttcttgtatgcatactccacagcacctggttattattcttggcgaaattcaaaggatggctcctggttcatccagtcgctttgtgccatgctgaaacagtatgccgacaagcttgaatttatgcacattcttacccgggttaaccgaaaggtggcaacagaatttgagtccttttcctttgacgctacttttcatgcaaagaaacagattccatgtattgtttccatgctcacaaaagaactctatttttatcactaactcgagatcgata Aminoacid sequence (SEQ ID NO: 464):DPSNMENTENSVDSKSIKNLEPKIIHGSESMDSISLDNSYKMDYPEMGLCIIINNKNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLIIQACRGTELDCGIETDSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFY H 118. 2e12scFv hIgG1 (SSS-P)H WCH2 WCH3-hcasp3-TM/CT (SEQ ID NO: 465) Nucleotidesequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccsatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggagaacactgaaaactcagtggattcaaaatccattaaaaatttggaaccaaagatcatacatggaagcgaatcaatggactctggaatatccctggacaacagttataaaatggattatcctgagatgggtttatgtataataattaataataagaattttcataaaagcactggaatgacatctcggtctggtacagatgtcgatgcagcaaacctcagggaaacattcagaaacttgaaatatgaagtcaggaataaaaatgatcttacacgtgaagaaattgtggaattgatgcgtgatgtttctaaagaagatcacagcaaaaggagcagttttgtttgtgtgcttctgagccatggtgaagaaggaataatttttggaacaaatggacctgttgacctgaaaaaaataacaaactttttcagaggggatcgttgtagaagtctaactggaaaacccaaacttttcattattcaggcctgccgtggtacagaactggactgtggcattgagacagacagtggtgttgatgatgacatggcgtgtcataaaataccagtggaggccgacttcttgtatgcatactccacagcacctggttattattcttggcgaaattcaaaggatggctcctggttcatccagtcgctttgtgccatgctgaaacagtatgccgacaagcttgaatttatgcacattcttacccgggttaaccgaaaggtggcaacagaatttgagtccttttcctttgacgctacttttcatgcaaagaaacagattccatgtattgtttccatgctcacaaaagaactctatttttatcactaactcgagatcgata Amino acid sequence (SEQ ID NO:466): MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKIKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSML TKBLYFYH 123.2e12 scFv hIgG1 (SSS-P)H P238SCH2 WCH3-hcasp3-TM/CT (SEQ ID NO: 467)Nucleotide sequence:aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggagaacactgaaaactcagtggattcaaaatccattaaaaatttggaaccaaagatcatacatggaagcgaatcaatggactctggaatatccctggacaacagttataaaatggattatcctgagatgggtttatgtataataattaataataagaattttcataaaagcactggaatgacatctcggtctggtacagatgtcgatgcagcaaacctcagggaaacattcagaaacttgaaatatgaagtcaggaataaaaatgatcttacacgtgaagaaattgtggaattgatgcgtgatgtttctaaagaagatcacagcaaaaggagcagttttgtttgtgtgcttctgagccatggtgaagaaggaataatttttggaacaaatggacctgttgacctgaaaaaaataacaaactttttcagaggggatcgttgtagaagtctaactggaaaacccaaacttttcattattcaggcctgccgtggtacagaactggactgtggcattgagacagacagtggtgttgatgatgacatggcgtgtcataaaataccagtggaggccgacttcttgtatgcatactccacagcacctggttattattcttggcgaaattcaaaggatggctcctggttcatccagtcgcttgccatgctgaaacagtatgccgacaagcttgaatttatgcacattcttacccgggttaaccgaaaggtggcaacagaatttgagtccttttcctttgacgctacttttcatgcaaagaaacagattccatgtattgtttccatgctcacaaaagaactctatttttatcactaactcgagatcgataa Amino acid sequence (SEQ ID NO:468): MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVIFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVBVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKIWLTREEIVELMRDVSKRDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSML TKELYFYH 124.hcasp8-TM/CT (SEQ ID NO: 469) hCaspase8B: Nucleotide sequence:ggatccttcgaacatggacttcagcagaaatctttatgatattggggaacaactggacagtgaagatctggcctccctcaagttcctgagcctggactacattccgcaaaggaagcaagaacccatcaaggatgccttgatgttattccagagactccaggaaaagagaatgttggaggaaagcaatctgtccttcctgaaggagctgctcttccgaattaatagactggatttgctgattacctacctaaacactagaaaggaggagatggaaagggaacttcagacaccaggcagggctcaaatttctgcctacagggtcatgctctatcagatttcagaagaagtgagcagatcagaattgaggtcttttaagtttcttttgcaagaggaaatctccaaatgcaaactggatgatgacatgaacctgctggatattttcatagagatggagaagagggtcatcctgggagaaggaaagttggacatcctgaaaagagtctgtgcccaaatcaacaagagcctgctgaagataatcaacgactatgaagaattcagcaaagagagaagcagcagccttgaaggaagtcctgatgaattttcaaatggggaggagtgtggggtaatgacaatctcggactctccaagagaacaggatagtgaatcacagactttggacaaagtttaccaaatgaaaagcaaacctcggggatactgtctgatcatcaacaatcacaattttgcaaaagcacgggagaaagtgcccaaacttcacagcattagggacaggaatggaacacacttggatgcaggggctttgaccacgacctttgaagagcttcattttgagatcaagccccacgatgactgcacagtagagcaaatctatgagattttgaaaatctaccaactcatggaccacagtaacatggactgcttcatctgctgtatcctctcccatggagacaagggcatcatctatggcactgatggacaggaggcccccatctatgagctgacatctcagttcactggtttgaagtgcccttcccttgctggaaaacccaaagtgttttttattcaggcttgtcagggggataactaccagaaaggtatacctgttgagactgattcagaggagcaaccctatttagaaatggatttatcatcacctcaaacgagatatatcccggatgaggctgactttctgctggggatggccactgtgaataactgtgtttcctaccgaaaccctgcagagggaacctggtacatccagtcactttgccagagcctgagagagcgatgtcctcgaggcgatgatattctcaccatcctgactgaagtgaactatgaagtaagcaacaaggatgacaagaaaaacatggggaaacagatgcctcagcctactttcacactaagaaaaaaacttgtcttcccttctgattgagcatgcatcgataAmino acid sequence (SEQ ID NO: 470):DPSNMDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYLNTRKLEEMERELQTPGRAQISAYRVMLYQISEEVSRSELRSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKRVCAQINKSLLKIINDYEEFSKERSSSLEGSPDEFSNGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHIDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMPQPTETLRKKLVFPSD hCaspase8C: Nucleotide sequence (SEQ ID NO:471):ggatccttcgaacatggacttcagcagaaatctttatgatattggggaacaactggacagtgaagatctggcctccctcaagttcctgagcctggactacattccgcaaaggaagcaagaacccatcaaggatgccttgatgttattattccagagactccaggaaagagaatgttggaggaaagcaatctgtccttcctgaaggagctgctcttccgaauaatagactggatttgctgattacctacctaaacactagaaaggaggagatggaaagggaacttcagacaccaggcagggctcaaatttctgcctacagggtcatgctctatcagatttcagaagaagtgagcagatcagaattgaggtcttttaagtttcttttgcaagaggaaatctccaaatgcaaactggatgatgacatgaacctgctggatattttcatagagatggagaagagggtcatcctgggagaaggaaagttggacatcctgaaaagagtctgtgcccaaatcaacaagagcctgctgaagataatcaacgactatgaagaattcagcaaaggggaggagttgtgtggggtaatgacaatctcggactctccaagagaacaggatagtgaatcacagactttggacaaagtttaccaaatgaaaagcaaacctcggggatactgtctgatcatcaacaatcacaattttgcaaaagcacgggagaaagtgcccaaacttcacagcattagggacaggaatggaacacacttggatgcaggggctttgaccacgacctttgaagagcttcattttgagatcaagccccacgatgactgcacagtagagcaaatctatgagattttgaaaatctaccaactcatggaccacagtaacatggactgcttcatctgctgtatcctctcccatggagacaagggcatcatctatggcactgatggacaggaggcccccatctatgagctgacatctcagttcactggtttgaagtgcccttcccttgctggaaaacccaaagtgttttttattcaggcttgtcagggggataactaccagaaaggtatacctgttgagactgattcagaggagcaaccctatttagaaatggatttatcatcacctcaaacgagatatatcccggatgaggctgactttctgctggggatggccactgtgaataactgtgttttcctaccgaaaccctgcagagggaacctggtacatccagtcactttgccagagcctgagagagcgatgtcctcgaggcgatgatattctcaccatcctgactgaagtgaactatgaagtaagcaacaaggatgacaagaaaaacatggggaaacagatgcctcagcctactttcacactaagaaaaaaacttgtcttcccttctgattgagcatgcatcgata Amino acid sequence (SEQ ID NO: 472):DPSNMDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYLNTRKEEMERELQTPGRAQISAYRVMLYQISEEVSRSELRSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKRVCAQINKSLLKIIDYEEFSKGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYTIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMP QPTFTLRKKLVFPSD125. 2e12 scFv hIgG1 (SSS-P)H WCH2 WCH3-hcasp8-TM/CT Nucleotide sequence(SEQ ID NO: 473):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggatcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctgggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtattttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcuccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacttcagcagaaatctttatgatattggggaacaactggacagtgaagatctggcctccctcaagtcctgagcctggactacattccgcacagcagaaatctttatgatattggggaacaactggacagtgaagatctggcctccctcaagttcctgagcctggactacattccgcatcctgaaggagctgctcttccgaattaatagactggatgctgattacctacctacctaaacactagaaaggaggagatggaaagggaacttcagacaccaggcagggctcaaatttctgcctacagggtcatgctctatcagaatttcagaagaagtgagcagatcagaattgaggtcttttaagtttcttgcaagaggaaatctccaaatgcaaactggatgatgacatgaacctgctggatattttcatagagatggagaagagggtcatcctgggagaaggaaagttggacatcctgaaaagagcttgcccaaatcaacaagagcctgctgaagataatcaacgactatgaagaattcagcaaagagaagcagcagccttgaaggaagtcctgatgaattttcaaatggggaggagttgtgtggggtaatgacaatctcggactctccaagagaacaggatagtgaatcacagactttggacaaagtttaccaaatgaaaagcaaacctcggggatactgtctgatcatcaacaatcacaattttgcaaaagcacgggagaaagtgcccaaacttcacagcattagggacaggaatggaacacacttggatgcaggggctttgaccacgacctttgaagagcttcattttgagatcaagccccacgatgactgcacagtagagcaaatctatgagattttgaaaatctaccaactcatggaccacagtaacatggactgcttcatctgcttcatcctctcccatggagacaagggcatcatctatggcactgatggacaggaggcccccatctatgagctgacatctcagttcactggtttgaagtgcccttcccttgctggaaaacccaaagtgttttttattcaggcttgtcagggggataactaccagaaaggtatacctgttgagactgattcagaggagcaaccctatttagaaatggatttatcatcacctcaaacgagatatatcccggatgaggctgacttttctgctggggatggccactgtgaataactgtgttttcctaccgaaaccctgcagagggaacctggtacatccagtcactttgccagagcctgagagagcgatgtcctcgaggcgatgatattctcaccatcctgactgaagtgaactatgaagcaagcaacaaggatgacaagaagaaaaacatggggaaaacagatgcctcagcctactttcacactaagaaaaaaacttgtcttcccttctgattgagcatgcatcgata Amino acid sequence(SEQ ID NO: 474):MFQVQWSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCPASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPLPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRLEESNLSFLKELLFRINRLDLLITYLNTRKEEMERELQTPGRAQISAYRVMLYQISEEVSRSELRSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKRVCAQINKSLLKIINDYEEFSKERSSSLEGSPDEFSNGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKMGKQMPQPTFTLRKKLVFPSD 126. 2e12 scFv hIgG1 (SSS-P)H P238SCH2WCH3-hcasp8B-TM/CT (other caspase 8 isoforms are similar) Nucleotidesequence (SEQ ID NO: 475):aagcttatggattttcaagtgcagatttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatgaaactgggtcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgttggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacttcagcagaaatctttatgatattggggaacaactggacagtgaagatctggcctccctcaagttcctgagcctggactacattccgcaaaggaagcaagaacccatcaaggatgccttgatgttattccagagactccaggaaaagagaatgttggaggaaagcaatctgtccttcctgaaggagctgctcttccgaattaatagactggatttgctgattacctacctaaacactagaaaggaggagatggaaagggaacttcagacaocaggcagggctcaaatttctgcctacagggtcatgctctatcagatttcagaagaagtgagcagatcagaattgaggtcttttaagtttcttttgcaagaggaaatctccaaatgcaaactggatgatgacatgaacctgctggatattttcatagagatggagaagagggtcatcctgggagaaggaaagttggacatcctgaaaagagtctgtgcccaaatcaacaagagcctgctgaagataatcaacgactatgaagaattcagcaaagagagaagcagcagccttgaaggaagtcctgatgaattttcaaatggggaggagttgtgtggggtaatgacaatctcggactctccaagagaacaggatagtgaatcacagactttggacaaagtttaccaaatgaaaagcaaacctcggggatactgtctgatcatcaacaatcacaattttgcaaaagcacgggagaaagtgcccaaacttcacagcattagggacaggaatggaacacacttggatgcaggggctttgaccacgacctttgaagagcttcattttgagatcaagccccacgatgactgcacagtagagcaaatctatgagattttgaaaatctaccaactcatggaccacagtaacatggactgcttcatctgctgtatcctctcccatggagacaagggcatcatctatggcactgatggacaggaggcccccatctatgagctgacatctcagttcactggtttgaagtgcccttcccttgctggaaaacccaaagtgttttttattcaggcttgtcagggggataactaccagaaaggtatacctgttgagactgattcagaggagcaaccctatttagaaatggatttatcatcacctcaaacgagatatatcccggatgaggctgactttctgctggggatggccactgtgaataactgtgtttcctaccgaaaccctgcagagggaacctggtacatccagtcactttgccagagcctgagagagcgatgtcctcgaggcgatgatattctcaccatcctgactgaagtgaactatgaagtaagcaacaaggatgacaagaaaaacatggggaaacagatgcctcagcctactttcacactaagaaaaaaacttgtcttcccttctgattgagcatgcatcgataa Amino acid sequence(SEQ ID NO: 476):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRWTFGGGTKLEIRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDFSRLYIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYLNTRKEEMERELQTPGRAQISAYRVMLYQISEEVSRSELSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKRVCAQINKSLLKIINDYEEFSKERSSSLEGSPDEFSNGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNCVSYRPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD 128. 2e12 scFv-hIgG1 (SSS-P)H WCH2WCH3-hFADD-TM/CT Nucleotide sequence (SEQ ID NO: 477):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaaatcaaacggggtggcggtgctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggaaactgggtcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtggccagagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtgcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacccgttcctggtgctgctgcactcggtgtcgtccagcctgtcgagcagcgagctgaccgagctcaagttcctatgcctcgggcgcgtggggcaagcgcaagctggagcgcgtgcagagcggcctagacctcttctccatgctgctggagcagaacgacctggagcccgggcacaccgagctcctgcgcgagctgctcgcctccctgcggcgccacgacctgctgctgcggcgcgtcgacgacttcgaggcgggggcggcggccggggccgcgcctggggaagaagacctgttgcagcatttaacgtcatatgtgataatgtggggaaagattggagaaggctggctcgtcagctcaaagtctcagacaccaagatcgacagcatcgaggacagatacccccgcaacctgacagagcgtgtgcgggagtcactgagaatctggaagaacacagagaaggagaacgcaacagtggcccacctggtgggggctctcaggtcctgccagatgaacctggtggctgacctggtacaagaggttcagcaggcccgtgacctccagaacaggagtggggccatgtccccgatgtcatggaactcagacgcatctacctccgaagcgtcctgataactcgagatcgataacaac Amino acid sequence(SEQ ID NO: 478):MDFQVQIFSFLLISASVASRGVDLVLTQSPASLAVSLGQATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHTELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQLKVSDTKIDSIEDRYPRNLTERVRESLRIWKNTEKENATVAHLVGALRSCQMNLVADLVQEVQQARDLQNRSGAMSPMSWNSDASTSEAS 129. 2e12 scFv-hIgG1(SSS-P)H P238SCH2 WCH3-hFADD-TM/CT Nucleotide sequence (SEQ ID NO: 479):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatgaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaagttttcttactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacccgttcctggtgctgctgcactcggtgtcgtccagcctgtcgagcagcgagctgaccgagctcaagttcctatgcctcgggcgcgtgggcaagcgcaagctggagcgcgtgcagagcggcctagacctcttctccatgctgctggagcagaacgacctggagcccgggcacaccgagctcctgcgcgagctgctcgcctccctgcggcgccacgacctgctgcggcgcgtcgacgacttcgaggcgggggcggcggccggggccgcgcctggggaagaagacctgtgtgcagcatttaacgtcatatgtgataatgtggggaaagattggagaaggctggctcgtcagctcaaagtctcagacaccaagatcgacagcatcgaggacagatacccccgcaacctgacagagcgtgtgcgggagtcactgagaatctggaagaacacagagaaggagaacgcaacagtggcccacctggtgggggctctcaggtcctgccagatgaacctggtggctgacctggtacaagaggttcagcaggcccgtgacctccagaacaggagtggggccatgtccccgatgtcatggaactcagacgcatctacctccgaagcgtcctgataactcgagatcgataacaac Amino acid sequence (SEQID NO: 480): MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVYSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNMVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEALHNHYTQKSLSLSPGKADPSNPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHTELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQLKVSDTKIDSIEDRYPRNLTERVRESLRIWKNTEKENATVAHLVGALRSCQMNLVADLVQEVQQARDLQNRSGAMSPMSWNSDASTSEAS 130.1D8 scFv hIgG1(SSS-P)H WCH2 WCH3-hCD80TM/CT Nucleotide sequence (SEQ ID NO: 481)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagccactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacacccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat Amino acid sequence (SEQ ID NO: 482):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSKLMDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKYNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 131.1D8 scFvmIgAT4-hCD80TM/CT Nucleotide sequence (SEQ ID NO: 483)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatcacatctgttctcctcctactactcctcctccaccttcctgccagcccagcctgtcactgcagcggccagctcttgaggacctgctcctgggttcagatgccagcatcacatgtactctgaatggcctgagagatcctgagggagctgtcttcacctgggagccctccactgggaaggatgcagtgcagaagaaagctgtgcagaattcctgcggctgctacagtgtgtccagcgtcctgcctggctgtgctgagcgctggaacagtggcgcatcattcaagtgcacagttacccatcctgagtctgacaccttaactggcacaattgccaaagtcacagtgaacaccttcccaccccaggtccacctgctaccgccgccgtcggaggagctggccctgaatgagctcgtgtccctgacatgcctggtgcgagctttcaaccctaaagaagtgctggtgcgatggctgcatggaaatgaggagctgtccccagaaagctacctagtgtttgagcccctaaaggagccaggcgagggagccaccacctacctggtgacaagcgtgttgcgtgtatcagctgaaatctggaaacagggtgaccagtactcctgcatggtgggccacgaggccttgcccatgaacttcacccagaagaccatcgaccgtctgtcgggtaaacccaccaatgtcagcgtgctgtgatcatgtcagagggagaggatccttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagaagggaaagtgtacgccctgtataaatcgatact Amino acid sequence (SEQ ID NO: 484):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDHICSPPTTPPPPSCQPSLSLQRPALEDLLLGSDASITCTLNGLRDPEGAVFTWEPSTGKDAVQKKAVQNSCGCYSVSSVLPGCAERWNSGASFKCTVTHPESDTLTGTIAKVTVNTFPPQVHLLPPPSEELALNELVSLTCLVRAFNPKEVLVRWLHGNEELSPESYLVFEPLKEPGEGATTYLVTSVLRVSAEIWKQGDQYSCMVGHEALPMNFTQKTIDRLSGKPTNVSVSVIMSEGEDPSNNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV 132.1D8 scFv hIgG1(SSS-P)H WCH2 WCH3-mFADD-TM/CT Nucleotide sequence (SEQ ID NO: 485)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattacgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacccattcctggtgctgctgcactcgctgtccggcagcctgtcgggcaacgatctgatggagctcaagttcttgtgccgcgagcgcgtgagcaaacgaaagctggagcgcgtgcagagtggcctggacctgttcacggtgctgctggagcagaacgacctggagcgcgggcacaccgggctgctgcgcgagttgctggcctcgctgcgccgacacgatctactgcagcgcctggacgacttcgaggcggggacggcgaccgctgcgcccccgggggaggcagatctgcaggtggcatttgacattgtgtgtgacaagtggggagagactggaaaagactggcccgcgagctgaaggtgtctgaggccaagatggatgggattgaggagaagtaccccgaagtctgagtgagcgggtaagggagagtctgaaagtctggaagaatgctgagaagaagaacgcctcggtggccggactggtcaaggcgctgcggacctgcaggctgaatctggtggctgacctggtggaagaagcccaggaatctgtgagcaagagtgagaatatgtccccagtactaagggattcaactgtgtcttcctcagaaacaccctgactcgagatcgatAmino acid sequence (SEQ ID NO: 486):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDQLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDPFLVLLHSLSGSLSGNDLMELKFLCRERVSKRKLERVQSGLDLFTVLLEQNDLERGHTGLLRELLASLRLLQRLDDFEAGTATAAPPGEADLQVAFDIVCDNVGRDWKRLARELKVSEAKMDGIEEKYPRSLSERVRESLKVWKNAEKKNASVAGLVKALRTCRLNLVADLVEEAQESVSKSENMSPVLRDSTVSSSETP 133.1D8 scFv hIgG1 (SSS-P)H P238SCH2 WCH3-mFADD-TM/CT Nucleotide sequence (SEQ ID NO: 487)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcncagtggcagtgggtctgggacctctattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaataatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgtgaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtgtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccgttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacccancctggtgctgctgcactcgctgtccggcagcctgtcgggcaacgatctgatggagctcaagttcttgtgccgcgagcgcgtgagcaaacgaaagctggagcgcgtgcagagtggcctggacctgttcacggtgctgctggagcagaacgacctggagcgcgggcacaccgggctgctgcgcgagttgctggcctcgctgcgccgacacgatctactgcagcgcctggacgacttcgaggcggggacggcgaccgctgcgcccccgggggaggcagatctgcaggtggcatttgacattgtgtgtgacaatgtggggagagactggaaaagactggcccgcgagctgaaggtgtctgaggccaagatggatgggattgaggagaagtacccccgaagtctgagtgagcgggtaagggagagtctgaaagtctggaagaatgctgagaagaagaacgcctcggtggccggactggtcaaggcgctgcggacctgcaggctgaatctggtggctgacctggtggaagaagcccaggaatctgtgagcaagagtgagaatatgtccccagtactaagggattcaactgtgtcttcctcagaaacaccctgactcgagatcgatAmino acid sequence (SEQ ID NO: 488):MDFQVQIFSFLLISASVIMSRGVDIYLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLETKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDPFLVLLHSLSGSLSGNDLMELKFLCRERVSKRKLERVQSGLDLFTVLLEQNDLERGHTGLLRELLASLRRRHDLLQRLDDFEAGTATAAPPGEADLQVAFDIVCDNVGRDWKRLARELKVSEAKMDGIEEKYPRSLSERVRESLKVWKNAEKKNASVAGLVKALRTCRLNLVADLVEEAQESVSKSENMSPVLRDSTVSSSETP 134.1D8 scFv hIgG1 (SSS-P)H WCH2WCH3-mcasp3-TM/CT Nucleotide sequence (SEQ ID NO: 489):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggagaacaacaaaacctcagtggattcaaaatccattaataattttgaagtaaagaccatacatgggagcaagtcagtggactctgggatctatctggacagtagttacaaaatggattatcctgaaatgggcatatgcataataattaataataagaacttccataagagcactggaatgtcatctcgctctggtacggatgtggacgcagccaacctcagagagacattcatgggcctgaaataccaagtcaggaataaaaatgatcttactcgtgaagacattttggaattaatggatagtgtttctaaggaagatcatagcaaaaggagcagctttgtgtgtgtgattctaagccatggtgatgaaggggtcatttatgggacaaatgggcctgttgaactgaaaaagttgactagcttcttcagaggcgactactgccggagtctgactggaaagccgaaactcttcatcattcaggcctgccggggtacggagctggactgtggcattgagacagacagtgggactgatgaggagatggcttgccagaagataccggtggaggctgacttcctgtatgcttactctacagcacctggttactattcctggagaaattcaaaggacgggtcgtggttcatccagtccctttgcagcatgctgaagctgtacgcgcacaagctagaatttatgcacattctcactcgcgttaacaggaaggtggcaacggaattcgagtccttctccctggactccactttccacgcaaagaaacagatcccgtgtattgtgtccatgctcacgaaagaactgtacttttatcactagctcgagatcgatg Amino acid sequence (SEQ ID NO: 490):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGKADPSNMENNKTSVDSKSINNFEVKTIHGSKSVDSGIYLDSSYKMDYPEMGICIIINNKNFHKSTGMSSRSGTDVDAANLRETFMGLKYQVRNKNDLTREDILELMDSVSKEDHSKRSSFVCVILSHGDEGVIYGTNGPVELKKLTSFFRGDYCRSLTGKPKLFIIQACRGTELDCGIETDSGTDEEMACQKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCSMLKLYAHKLEFMHILTRVNRKVATEFESFSLDSTFHAKKQIPCIVSMLTKELYFYH 135.1D8 scFv hIgG1(SSS-P)H P238S CH2 WCH3-mcasp3-TM/CT Nucleotide sequence (SEQ ID NO:491):Aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgaccacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggscctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctga Amino acid sequence (SEQ ID NO: 492):MDFQVQIFSFLLISASVIMSRGVDIVLTTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNSNMENNKTSVDSKSINNFEVKTIHGSKSVDSGIYLDSSYKMDYTPEMGICIIINNKNFHKSTGMSSRSGTDVDAANLRETFMGLKYQVRNKNDLTREDILELMDSVSKEDHSKRSSFVCVIILSHGDEGVIYGTNGPVELKKLTSFFRGDYCRSLTGKPKLFIIQACRGTELDCGIETDSGTDEEMACQKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCSMLKLYAHKLEFMHILTRVNRKVATEFESFSLDSTFHAKKQIPCIVSMLTKELYFYH 136.1D8 scFv hIgG1(SSS-P)H WCH2 WCH3-mcasp8-TM/CT Nucleotide sequence (SEQ ID NO: 493):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggatttccagagttgtctttatgctattgctgaagaactgggcagtgaagacctggctgccctcaagttcctgtgcttggactacatcccacacaagaagcaggagaccatcgaggatgcccagaagctatttctgaggctgcgggaaaaggggatgttggaggaaggcaatctgtctttcctgaaagagctgcttttccacatcagtcggtgggacctgctggtcaacttcctagactgcaaccgagaggagatggtgagagagctgcgggatccagacaatgcccagatttctccctacagggtcatgctctttaagctctcagaagaagtgagcgagttggaattgagatcttttaagttccttttgaacaatgagatccccaaatgtaagctggaagatgacttgagcctgcttgaaatttttgtagaaatggagaagaggaccatgctggcagaaaataacttggaaaccctaaaatcaatctgtgaccaggtcaacaagagcctgctggggaagatcgaggattatgaaagatcaagcacagagagaagaatgagccttgaagtgagtcacggacttcagacaaagtttaccaaatgaagaacaaacctcggggatactgtctgatcatcaacaatcatgatttcagcaaggcccgggaagacataacccaactccgaaaaatgaaggacagaaaaggaacagactgtgataaagaggctctgagtaagacctttaaggagcttcattttgagatagtatcttacgacgactgcactgcaaatgaaatccacgagattctagaaggctaccaaagcgcagaccacaagaacaaagactgcttcatctgctgtatcctatcccacggtgacaagggtgtcgtctatggaacggatgggaaggaggcctccatctatgacctgacatcttacttcactggttcaaagtgcccttccctgtctgggaaacccaagatctttttcattcaggcttgccaaggagtaacttccagaaaggagtgcctgatgaggcaggcttcgagcaacagaaccacactttagaagtggattcatcatctcacaagaactatattccggatgaggcagactttctgctgggaatggctacggtgaagaactgcgtttcctaccgagatcctgtgaatggaacctggtatattcagtcactttgccagagcctgagggaaagatgtcctcaaggagatgacattcttagcatcctgactggcgtgaactatgacgtgagcaataaagacgacaggaggaacaagggaaagcagatgccacagcccaccttcacactacggaagaagctcttcttccctccctaatgactcgagatcgatt Amino acid sequence (SEQ ID NO: 494):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDFQSCLYAIAEELGSEDLAALKFLCLDYIPHKKQETIEDAQKLFLRLREKGMLEEGNLSFLKELLFHISRWDLLVNFLDCNREEMVRELRDPDNAQISPYRVMLFKLSEEVSELELRSFKFLLNNEIPKCKLEDDLSLLEIFVEMEKRTMLAENNLETLKSICDQVNKSLLGKIEDYERSSTERRMSLEGREELPPSVLDEMSLKMAELCDSPREQDSESRTSDKVYQMKNKPRGYCLIINNHDFSKAREDITQLRKMKDRKGTDCDKEALSKTFKELHFEIVSYDDCTANEIHEILEGYQSADHKNKDCFICCILSHGDKGVVYGTDGKEASIYDLTSYFTGSKCPSLSGKPKIFFIQACQGSNFQKGVPDEAGFEQQNHTLEVDSSSHKNYIPDEADFLLGMATVKNCVSYRDPVNGTWTIQSLCQSLRERCPQGDDILSILTGVNYDVSNKDDRRNKGKQMPQPTFTLRKKLFFPP 137.1D8 scFv hIgG1 (SSS-P)H P238SCH2WCH3-mcasp8-TM/CT Nucleotide sequence (SEQ ID NO: 495):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttatactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggatttccagagttgtctttatgctattgctgaagaactgggcagtgaagacctggctgccctcaagttcctgtgcttggactacatcccacacaagaagcaggagaccatcgaggatgcccagaagctatttctgaggctgcgggaaaaggggatttggaggaaggcaatctgtctttcctgaaagagctgcttttccacatcagtcggtgggacctgctggtcaacttcctagactgcaaccgagaggagatggtgagagagctgcgggatccagacaatgcccagatttctccctacagggtcatgctctttaagctctcagaagaagtgagcgagttggaattgagatcttttaagttccttttgaacaatgagatccccaaatgtaagctggaagatgacttgagcctgcttgaaatttttgtagaaatggagaagaggaccatgctggcagaaaataacttggaaaccctaaaatcaatctgtgaccaggtcaacaagagcctgctggggaagatcgaggattatgaaagatcaagcacagagagaagaatgagccttgaaggaagggaagagttgccaccttcagtggatgagatgagcctcaaaatggcggaactggactcgccaagagaacaagacagtgagtcacggacttcagacaaagtttaccaaatgaagaacaaacctcggggatactgtctgatcatcaacaatcatgatttcagcaaggcccgggaagacataacccaactccgaaaaatgaaggacagaaaaggaacagactgtgataaagaggctctgagtaagacctttaaggagcttcattttgagatagtatcttacgacgactgcactgcaaatgaaatccacgagattctagaaggctaccaaagcgcagaccacaagaacaaagactgcttcatctgctgtatcctatcccacggtgacaagggtgtcgtctatggaacggatgggaaggaggcctccatctatgacctgacatcttacttcactggttcaaagtgcccttccctgtctgggaaacccaagatctttttcattcaggcttgccaaggaagtaacttccagaaaggagtgcctgatgaggcaggcttcgagcaacagaaccacactttagaagtggattcatcatctcacaagaactatattccggatgaggcagactttctgctgggaatggctacggtgaagaactgcgtttcctaccgagatcctgtgaatggaacctggtatattcagtcactttgccagagcctgagggaaagatgtcctcaaggagatgacattcttagcatcctgactggcgtgaactatgacgtgagcaataaagacgacaggaggaacaagggaaagcagatgccacagcccaccttcacactacggaagaagctcttcttccctccctaatgactcgagatcgattc Amino acid sequence (SEQ ID NO: 496):MDFQVQIFSFLLISASVIMSRGVDILVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEALHNHYTQKSLSLSPGKADPSNMDFQSCLYAIAEELGSEDLAALKFLCLDYIPHKKQETIEDAQKLFRLRELREKGMLEEGNLSFLKELLFHISRWDLLVNFLDCNREEMVRELRDPDNAQISPYRVMLFKLSEEVSELELRSFKFLLNNEIPKCKLEDDLSLLEIFVEMEKRTMLAENNLETLKSICDQVNKSLLGKIEDYERSSTERRMSLEGREELPPSVLDEMSLKMAELCDSPREQDSESRTSDKVYQMKNKPRGYCLIINNHDFSKAREDITQLRKMKDRKGTDCDKEALSKTFKELHYEIVSYDDCTANEIHEILEGYQSADHKNKDCFICCILSHGDKGVVYGTDGKEASIYDLTSYFTGSKCPSLSGKPKIFFIQACQGSNFQKGVPDEAGFEQQNHTLEVDSSSHKNYIPDEADFLLGMATVKNCVSYRDPVNGTWYIQSLCQSLRERCPQGDDILSILTGVNYDVSNKDDRRNKGKQMPQPTFTLRKKLFFPP 138.1D8 scFv hIgG1 (SSS-P)H WCH2WCH3-hcasp3-TM/CT (SEQ ID NO: 497)aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcggcggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaatcaacaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgtgaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcag˜gagcctctccctgtctccgggaagcggatccttcgaacatggagaacactgaaaactcagtggattcaaaatccattaaaaatttggaaccaaagatcatacatggaagcgaatcaatggactctggaatatccctggacaacagttataaaatggattatcctgagatgggtttatgtataataattaataataagaattttcataaaagcactggaatgacatctcggtctggtacagatgtcgatgcagcaaacctcagggaaacattcagaaacttgaaatatgaagtcaggaataaaaatgatcttacacgtgaagaaatttggaattgatgcgtgatgtttctaaagaagatcacagcaaaaggagcagttttgtttgtgtgcttctgagccatggtgaagaaggaataattttggaacaaatggacctgttgacctgaaaaaaataacaaactttttcagaggggatcgttgtagaagtctaactggaaaacccaaacttttcattattcaggcctgccgtggtacagaactggactgtggcattgagacagacagtggtgttgatgatgacatggcgtgtcataaaataccagtggaggccgacttcttgtatgcatactccacagcacctggttattattcttggcgaaattcaaaggatggctcctggttcatccagtcgctttgtgccatgctgaaacagtatgccgacaagcttgaatttatgcacattcttacccgggttaaccgaaaggtggcaacagaatttgagtccttttcctttgacgctacttttcatgcaaagaaacagattccatgtattgtttccatgctcacaaaagaactctatttttatcactaactcgagatcgataAmino acid sequence (SEQ ID NO: 498):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWTYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVNTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH 139.1D8 scFv hIgG1(SSS-P)H P238SCH2 WCH3-hcasp3-TM/CT Nucleotide sequence (SEQ ID NO:499):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcucctcuccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaagcggatccttcgaacatggagaacactgaaaactcagtggattcaaaatccattaaaaatttggaaccaaagatcatacatggaagcgaatcaatggactctggaatatccctggacaacagttataaaatggattatcctgagatgggtttatgtataataattaataataagaattttcataaaagcactggaatgacatctcggtctggtacagatgtcgatgcagcaaacctcagggaaacattcagaaacttgaaatatgaagtcaggaataaaaatgatcttacacgtgaagaaattgtggaattgatgcgtgatgtttctaaagaagatcacagcaaaaggagcagttttgtttgtgtgcttctgagccatggtgaagaaggaataatttttggaacaaatggacctgttgacctgaaaaaaataacaaactttttcagaggggatcgttgtagaagtctaactggaaaacccaaacttttcattattcaggcctgccgtggtacagaactggactgtggcattgagacagacagtggtgttgatgatgacatggcgtgtcataaaataccagtggaggccgacttcttgtatgcatactccacagcacctggttattattcttggcgaaattcaaaggatggctcctggttcatccagtcgctttgtgccatgctgaaacagtatgccgacaagcttgaatttatgcacattcttacccgggttaaccgaaaggtggcaacagaatttgagtccttttcctttgacgctacttttcatgcaaagaaacagattccatgtattgtttccatgctcacaaaagaactctatttttatcactaactcgagatcgataaAmino acid sequence (SEQ ID NO: 500):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMTSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKYREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMENTENSVDSKSIKNLEPKIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVDDDMACHTKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQLPCIVSMLTKELYFYH 140.1D8 scFv hIgG1(SSS-S)H WCH2 WCH3-hcasp8-TM/CT Nucleotide sequence (SEQ ID NO: 501):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacttcagcagaaatctttatgatattggggaacaactggacagtgaagatctggcctccctcaagttcctgagcctggactacattccgcaaaggaagcaagaacccatcaaggatgccttgatgttattccagagactccaggaaaagagaatgttggaggaaagcaatctgtccttcctgaaggagctgctcttccgaattaatagactggatttgctgattacctacctaaacactagaaaggaggagatggaaagggaacttcagacaccaggcagggctcaaatttctgcctgacagggtcatgctctatcagatttcagaagaagtgagcagatcagaattgaggtcttttaagtttcttttgcaagaggaaatctccaaatgcaaactggatgatgacatgaacctgctggatattttcatagagatggagaagagggtcatcctgggagaaggaaagttggacatcctgaaaagagtctgtgcccaaatcaacaagagcctgctgaagataatcaacgactatgaagaattcagcaaagagagaagcagcagccttgaaggaagtcctgatgaattttcaaatggggaggagttgtgtggggtaatgacaatctcggactctccaagagaacaggatagtgaatcacagactttggacaaagtttaccaaatgaaaagcaaacctcggggatactgtctgatcatcaacaatcacaattttgcaaaagcacgggagaaagtgcccaaacttcacagcattagggacaggaatggaacacacttggatgcaggggctttgaccacgacctttgaagagcttcattttgagatcaagccccacgatgactgcacagtagagcaaatctatgagattttgaaaatctaccaactcatggaccacagtaacatggactgcttcatctgctgtatcctctcccatggagacaagggcatcatctatggcactgatggacaggaggcccccatctatgagctgacatctcagttcactggtttgaagtgcccttcccttcccttgctggaaaacccaaagtgttttttattcaggcttgtcagggggataactaccagaaaggtatacctgttgagactgattcagaggagcaaccctatttagaaatggatttatcatcacctcaacgagatatatcccggatgaggctgactttctgctggggatggccactgtgaataactgtgtttcctaccgaaaccctgcagagggaacctggtacatccagtcactttgccagagcctgagagagcgatgtcctcgaggcgatgatattctcaccatcctgactgaagtgaactatgaagtaagcaacaaggatgacaagaaaaacatggggaaacagatgcctcagcctactttcacactaagaaaaaaacttgtcttcccttctgattgagcatgcatcgata Amino acid sequence (SEQ ID NO: 502):MSFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSVSYMYWYQQKSGASPKLWIYDTSKLASQVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSIRDTSKSTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTEVTCVVVDDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKADPSNMDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYLNTRKEEMERELQTPGRAQISAYRVMLYQISEEVSRSELRSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGRGKLDILKRVAQINKSLLKIINDYEEFSKERSSSLEGSPDEFSNGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEDFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD 141.1D8 scFv hIgG1 (SSS-S)H P238SCH2WCH3-hcasp8-TM/CT Nucleotide sequence (SEQ ID NO: 503):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggcggtggtgggtcgggtcgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctgcaaccgacacagaccctgtccctcacatgcactgtctctggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatgggaataatatattatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagttttcttaaaaatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagtcatggtcacagtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacatggacttcagcagaaatctttatgatattggggaacaactggacagtgaagatctggcctccctcaagttcctgagcctggactacattccgcaaaggaagcaagaacccatcaaggatgccttgatgttattccagagactccaggaaaagagaatgttggaggaaagcaatctgtccttcctgaaggagctgctcttccgaattaatagactggatttgctgattacctacctaaacactagaaaggaggagatggaaagggaacttcagacaccaggcagggctcaaatttctgcctacagggtcatgctctatcagatttcagaagaagtgagcagatcagaattgaggtcttttaagtttcttttgcaagaggaaatctccaaatgcaaactggatgatgacatgaacctgctggatattttcatagagatggagaagagggtcatcctgggagaaggaaagttggacatcctgaaaagagtctgtgcccaaatcaacaagagcctgctgaagataatcaacgactatgaagaattcagcaaagagagaagcagcagccttgaaggaagtcctgatgaattttcaaatggggaggagttgtgtggggtaatgacaatctcggactctccaagagaacaggatagtgaatcacagactttggacaaagtttaccaaatgaaaagcaaacctcggggatactgtctgatcatcaacaatcacaattttgcaaaagcacgggagaaaggcccaaacttcacagcattagggacaggaatggaacacacttggatgcaggggctttgaccacgacctttgaagagcttcattttgagatcaagccccacgatgactgcacagtagagcaaaatctatgagattttgaaaatctaccaactcatggaccacagtaacatggactgcttcatctgctgtatcctctcccatggagacaagggcatcatctatggcactgatggacaggaggcccccatctatgagctgacatctcagttcactggtttgaagtgcccttcccttgctggaaaacccaaagtgttttttattcaggcttgtcagggggataactaccagaaaggtatacctgttgagactgattcagaggagcaaccctatttagaaatggatttatcatcacctcaacgacgagatatatcccggatgaggctgactttctgctggggatggccactgtgaataactgtgtttcctaccgaaaccctgcagagggaacctggtcatccagtcactttgccagagcctgagagagcgatgtcctcgaggcgatgatattctcaccatcctgactgaagtgaactatgaagtaagcaacaaggatgacaagaaaaacatggggaaacagatgcctcagcctactttcacactaagaaaaaaacttgtcttcccttctgattgagcatgcatcgataa Amino acid sequence (SEQ ID NO: 504):MDFQVQIFSFLLISASVIMSRGVDIVLTQSPTTIAASPGEKVTITCRASSSVSYMYWYQQKSGASPKLWIYDTSKLASGVPNRFSGSGSGTSYSLAINTMETEDAATYYCQQWSSTPLTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLKEAGPGLVQPTQTLSLTCTVSGFSLTSDGVHWIRQPPGKGLEWMGIIYYDGGTDYNSAIKSRLSISRDTSKSQVFLKINSLQTDDTAMYYCARIHFDYWGQGVMVTVSSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNMDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYLNTRKEEMERELQTPGRAQISAYRVMLYQISEEVSRSELRSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKIDILKRVCAQINKSLLKIINDYEEFSKERSSSLEGSPDEFSNGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEIILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD 145. HCTLA4 IgAH IgACH2CH3 Nucleotide sequence(SEQ ID NO: 505):atggcttgccttggatttcagcggcacaaggctcagctgaacctggctgccaggacctggccctgcactctcctgttttttcttctcTTcatccctgtcttctgcaaagcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgacggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggacggcacctgctactgataatctaga Amino acidsequence (SEQ ID NO: 506):MACLGFQRHKAQLNLAARTWPCTLLFFLLFIPVFCKAMHVAQPAVVYLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMTGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCALEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSGMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY 146. hCTLA4 IgA WH WCH2 T4CH3 (hCTLA4 IgAHIgACH2CH3) Nucleotide sequence (SEQ ID NO: 507):atggcttgccttggatttcagcggcacaaggctcagctgaacctggctgccaggacctggccctgcactctcctgtttttcttctcttcatccctgtcttctgcaaagcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacctacatgacggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggaggtggactgataatctaga Amino acid sequence (SEQID NO: 508): MACLGFQRHKAQLNLAARTWPCTLLFFLLFIPVFCKAMHVAQPAVVTLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMTGNELTFLDDSICTGTSSGNQVNLTIQGLRMADTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVD 147. hIGA WH WCH2 T18CH3 Nucleotide sequence (SEQ IDNO: 509):Tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaatgataatctaga Amino acid sequence (SEQ IDNO: 510): DQPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEAILTCTLTGLRDASGVTFTWTPSSGKSAVQGPPDRDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAF TQKTIDRLAGK 150.G19-4 scFv (SSS-P)WH WCH2 WCH3-hCD80TMCT Nucleotide sequence (SEQ ID NO:511):aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgggagctcggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctatcgatgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgtcctgcaaggcctcggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat Amino acid sequence (SEQID NO: 512): MDFQVQIFSFLLISASVIMSRGVDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRELGGGGSGGGGSGGGGSIDEVQLQQSGPELVKPGASMSCKASGYSFTGYIVNWLKQSHGKNLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSDLEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVR PVHCD16lowFL + NL Nucleotide sequence (SEQ ID NO: 513):aagcttgccgccatgtggcagctgctcctcccaactgctctgctacttctagtttcagctggcatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggcttgttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggcagtgtcaaccatctcatcattctttccacctgggtaccaagtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaatgacccAmino acid sequence (SEQ ID NO: 514):MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRXDPQDK

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Accordingly, the presentinvention is not limited except as by the appended claims.

All patents, patent applications, publications, scientific articles, websites, and other documents and materials referenced or mentioned hereinare indicative of the levels of skill of those skilled in the art towhich the invention pertains, and each such referenced document andmaterial is hereby incorporated by reference to the same extent as if ithad been incorporated by reference in its entirety individually or setforth herein in its entirety. Additionally, all claims in thisapplication, and all priority applications, including but not limited tooriginal claims, are hereby incorporated in their entirety into, andform a part of, the written description of the invention. Applicantsreserve the right to physically incorporate into this specification anyand all materials and information from any such patents, applications,publications, scientific articles, web sites, electronically availableinformation, and other referenced materials or documents. Applicantsreserve the right to physically incorporate into any part of thisdocument, including any part of the written description, the claimsreferred to above including but not limited to any original claims.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of may be replaced with either of the other two terms in thespecification. Also, the terms “comprising”, “including”, containing”,etc. are to be read expansively and without limitation. The methods andprocesses illustratively described herein suitably may be practiced indiffering orders of steps, and that they are not necessarily restrictedto the orders of steps indicated herein or in the claims. It is alsothat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, a reference to “a host cell”includes a plurality (for example, a culture or population) of such hostcells, and so forth. Under no circumstances may the patent beinterpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. For example, thesubject matter of the instant invention may optionally exclude any ofthe subject matter or sequences described or included in relatedapplication published as U.S.S.N. 2003/0118592 A1 on Jun. 26, 2003 (andthe sequence listings of SEQ ID NOS: 1-427 published by USPTO), byLedbetter et al. entitled “Binding Domain-Immunoglobulin FusionProteins”.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A fusion protein that binds CD20 comprising an amino acid sequence asset forth in: SEQ ID NO:135 (2H7 scFv (CSS-S)H WCH2 WCH3), whereinproline at position 283 is substituted with serine; SEQ ID NO:137 (2H7scFv (SCS-S)H WCH2 WCH3), wherein proline at position 283 is substitutedwith serine; SEQ ID NO:166 (2H7 scFv (CSC-S)H WCH2 WCH3), whereinproline at position 283 is substituted with serine; SEQ ID NO:372 (2H7scFv VHL11S (CSS-S)H WCH2 WCH3); SEQ ID NO:246 (2H7 scFv VH L11S(CSC-S)H WCH2 WCH3); SEQ ID NO:370 (2H7 scFv VH L11S (SSS-S)H WCH2WCH3); SEQ ID NO:268 (2H7 scFv VH L11S (CSS-S)H K322S CH2 WCH3); or SEQID NO:276 (2H7 scFv VH L11S (CSS-S)H P331S CH2 WCH3).
 2. A fusionprotein that binds CD20 consisting essentially of an amino acid sequenceas set forth in SEQ ID NO:135 (2H7 scFv (CSS-S)H WCH2 WCH3) whereinproline at position 283 is substituted with serine, SEQ ID NO:166 (2H7scFv (CSC-S)H WCH2 WCH3) wherein proline at position 283 is substitutedwith serine, SEQ ID NO:372 (2H7 scFv VHL11S (CSS-S)H WCH2 WCH3), or SEQID NO:246 (2H7 scFv VH L11S (CSC-S)H WCH2 WCH3).
 3. A fusion proteincomprising from amino-terminus to carboxy-terminus: (i) an scFv bindingdomain polypeptide that binds CD20, wherein the scFv comprises aminoacids 23-265 of SEQ ID NO:246; (ii) an immunoglobulin hinge polypeptideconsisting of amino acids 269-283 as set forth in SEQ ID NO:246; and(iii) an amino-terminally truncated immunoglobulin heavy chain constantregion polypeptide consisting of amino acids 284-500 as set forth in SEQID NO:246.
 4. A pharmaceutical composition comprising a fusion proteinaccording to claim 1 and a physiologically acceptable carrier.
 5. Apharmaceutical composition comprising a fusion protein according toclaim 2 and a physiologically acceptable carrier.
 6. A pharmaceuticalcomposition comprising a fusion protein according to claim 3 and aphysiologically acceptable carrier.